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Health Evaluation of Experimental Laboratory Mice
Abstract
Good science and good animal care go hand in hand. A sick or distressed animal does not produce the reliable results that a healthy and unstressed animal produces. This unit describes the essentials of assessing mouse health, colony health surveillance, common conditions, and determination of appropriate endpoints. Understanding the health and well-being of the mice used in research enables the investigator to optimize research results and animal care. Curr. Protoc. Mouse Biol. 2:145-165 © 2012 by John Wiley & Sons, Inc.
... Secondly, we use a combination of cyanoacrylate tissue adhesive and UV light-curing resin, which significantly reduces surgery time, improves wound healing, and reduces the postoperative recovery period, with a near 100% success rate. Thirdly, we have designed a customized welfare assessment scoresheet to monitor animals undergoing long-term cannula implantation, including indicators that accurately and effectively reflect animals' well-being for this particular surgery [15][16][17][18][19][20][21][22][23]. By implementing all the improvements, we evidenced a positive impact on animal welfare, the reduction in the number of animals used, and the quality of the experimental data, all of which align with the "refinement" and "reduction" principles of the 3Rs [13]. ...
... and A.C.-V. or J.C.-S.; an example of the "postoperative monitoring form" is provided in Supplementary Information S3). To ensure consistency and reproducibility, a scoresheet was designed, including several indicators related to general condition, nutritional and hydration status, spontaneous behavior, and surgery-related parameters, as well as compensatory measures, as detailed in Table 4 [15,17,[20][21][22]39,40]. Overall, these welfare indicators were carefully chosen based on our experience with these specific surgical procedures and their relevance, ease of recognition, reliability, and effectiveness in providing accurate assessment of welfare [17]. ...
... [39,40], among others). In our study, we designed a standardized scoresheet for effective and accurate follow-up of the animal's overall well-being, covering a wide range of indicators related to general condition, physical appearance and posture, nutritional and hydration status, spontaneous behavior, and the surgical procedure itself [15][16][17][18][19][20][21][22][23]. Our results corroborate the fact that relying on a single parameter is insufficient in most cases. ...
Stereotaxic surgeries enable precise access to specific brain regions, being of particular interest for chronic intracerebroventricular drug delivery. However, the challenge of long-term studies at this level is to allow the implantation of drug storage devices and their correct intrathecal connection while guaranteeing animal welfare during the entire study period. In this study, we propose an optimized method for safe intrathecal device implantation, focusing on preoperative, intraoperative, and postoperative procedures, following the 3Rs principle and animal welfare regulations. Our optimized protocol introduces three main refinements. Firstly, we modify the dimensions of the implantable devices, notably diminishing the device-to-mouse weight ratio. Secondly, we use a combination of cyanoacrylate tissue adhesive and UV light-curing resin, which decreases surgery time, improves healing, and notably minimizes cannula detachment or adverse effects. Thirdly, we develop a customized welfare assessment scoresheet to accurately monitor animal well-being during long-term implantations. Taken together, these refinements positively impacted animal welfare by minimizing the negative effects on body weight, surgery-related complications, and anxiety-like behaviors. Overall, the proposed refinements have the potential to reduce animal use, enhance experimental data quality, and improve reproducibility. Additionally, these improvements can be extended to other neurosurgical techniques, thereby advancing neuroscience research, and benefiting the scientific community.
... No alteration in the general health status of stressed animals emerged at the end of the stress paradigm. The health measures were taken by the animal caretakers through the daily observation of the animals in their home cages in order to assess both behavioral and physical indicators of welfare [79]. These included hunched posture, dull or sluggish movements, reduced locomotion/immobility, altered nest building and stereotypic behaviors, excessive grooming, absence of feces, rough hair coat, squinted eyes and skin abrasions/lesions [79]. ...
... The health measures were taken by the animal caretakers through the daily observation of the animals in their home cages in order to assess both behavioral and physical indicators of welfare [79]. These included hunched posture, dull or sluggish movements, reduced locomotion/immobility, altered nest building and stereotypic behaviors, excessive grooming, absence of feces, rough hair coat, squinted eyes and skin abrasions/lesions [79]. ...
Fragile X syndrome (FXS) is a pervasive developmental disorder and the most common monogenic cause of autism spectrum disorder (ASD). Female heterozygous (HET) carriers play a major role in the transmission of the pathology and present several FXS- and ASD-like behavioral alterations. Despite their clear genetic origins, FXS symptoms are known to be modulated by environmental factors, e.g., exposure to chronic stress, especially during critical life periods, such as pregnancy. Pregnancy, together with pups' care, constitutes maternal experience, i.e., another powerful environmental factor affecting several neurobehavioral functions in females. Here we investigated the impact of maternal experience on the long-term effects of stress in Fmr1-HET female mice. Our findings demonstrated that the behavioral abnormalities of HET females, i.e., hyperactivity and memory deficits, were unaffected by stress or maternal experience. In contrast, stress, independently of maternal experience, induced the appearance of cognitive deficits in WT mice. Maternal experience increased anxiety levels in all mice and enhanced their corticosterone levels, concomitantly promoting the effects of stress on social communication and adrenal glands. In translational terms, these results advance our understanding of the environmental modulation of the behavioral alterations observed in FXS female carriers and highlight the long-term impact of maternal experience and its interactions with chronic stress.
... No alteration in the general health status of stressed animals emerged at the end of the stress paradigm. The health measures were taken by the animal caretakers through the daily observation of the animals in their home cage in order to assess both behavioral and physical indicators of welfare [76]. These included hunched posture, dull or sluggish movements, reduced lo-10 of 17 comotion/immobility, altered nest building and stereotypic behaviors, excessive grooming, absence of feces, rough hair coat, squinted eyes and skin abrasions/lesions [76]. ...
... The health measures were taken by the animal caretakers through the daily observation of the animals in their home cage in order to assess both behavioral and physical indicators of welfare [76]. These included hunched posture, dull or sluggish movements, reduced lo-10 of 17 comotion/immobility, altered nest building and stereotypic behaviors, excessive grooming, absence of feces, rough hair coat, squinted eyes and skin abrasions/lesions [76]. ...
Fragile X Syndrome (FXS) is a pervasive developmental disorder and the most common monogenic cause of autism spectrum disorder (ASD). Female heterozygous (HET) carriers play a major role in the transmission of the pathology and present several FXS- and ASD-like behavioral alterations. Despite their clear genetic origins, FXS symptoms are known to be modulated by environmental factors, e.g., the exposure to chronic stress, especially during critical life periods, such as pregnancy. Pregnancy, together with pups’ care, constitutes maternal experience, i.e., another powerful environmental factor affecting several neurobehavioral functions in females. Here we investigated the impact of maternal experience on the long-term effects of stress in Fmr1-HET female mice. Our findings demonstrated that the behavioral abnormalities of HET females, i.e., hyperactivity and memory deficits, were unaffected by stress or maternal experience. In contrast, stress, independently of maternal experience, induced the appearance of cognitive deficits in WT mice. Maternal experience increased anxiety levels in all mice and enhanced their corticosterone levels, concomitantly promoting the effects of stress on social communication and adrenal glands. Our results advance our understanding of the environmental modulation of the behavioral alterations observed in FXS mice and highlight the long-term impact of maternal experience and its interactions with chronic stress.
... In addition, G3 and G4 received 1.5% (w/v) DSS (DSS for colitis, TdB Consultancy, Sweden, MW 40,000) in drinking water for 9 consecutive days (15-23) to induce colitis, followed by 15 days of washout (days [24][25][26][27][28][29][30][31][32][33][34][35][36][37][38]. Fresh DSS solution was prepared every day and administered to mice. ...
... As shown in Table 1, a specific pain scale was used in order to minimize animal pain and distress. Different parameters such as general appearance, food/water intake, behavior, nest building, were assessed as indicators of health and welfare in mice (36)(37)(38)(39). Body weight was measured weekly for mice of G1 and G2 throughout the whole experimental period and daily during the DSS treatment period for mice of G3 and G4. ...
Introduction:
Inflammatory bowel diseases (IBD) are chronic inflammatory conditions that typically involve diarrhea, abdominal pain, fatigue, and weight loss, with a dramatic impact on patients' quality of life. Standard medications are often associated with adverse side effects. Thus, alternative treatments such as probiotics are of great interest. The purpose of the present study was to evaluate the effects of oral administration of Lentilactobacillus kefiri (basonym: Lactobacillus kefiri) SGL 13 and Andrographis paniculata, namely, Paniculin 13™, on dextran sodium sulfate (DSS)- treated C57BL/6J mice.
Methods:
Colitis was induced by administering 1.5% DSS in drinking water for 9 days. Forty male mice were divided into four groups, receiving PBS (control), 1.5% DSS, Paniculin 13™ and 1.5% DSS + Paniculin 13™.
Results:
The results showed that body weight loss and Disease Activity Index (DAI) score were improved by Paniculin 13™. Moreover, Paniculin 13™ ameliorated DSS-induced dysbiosis, by modulating the gut microbiota composition. The gene expression of MPO, TNFα and iNOS in colon tissue was reduced and these data matched with the histological results, supporting the efficacy of Paniculin 13™ in reducing the inflammatory response. No adverse effects were associated to Paniculin 13™ administration.
Discussion:
In conclusion, Paniculin 13™ could be an effective add-on approach to conventional therapies for IBD.
... Females have a higher fat distribution than males [13,14]. The exclusion criterion was adult female Mus musculus which have abnormalities, such as gait abnormalities, tumors in body organs, and pale, red, or bluish color in the mucous membrane around the eyes [15]. ...
... In advance decay stage, the degree of degradation is complete, body looks dry, and dark in color. The dry/remains stage is characterized the organs are gone and it is odorless [3,15]. ...
Anatomy studies require cadavers to study the human body. Generally in Indonesia, the dead human body will be buried. This causes problems because the decomposition process of a cadaver that is preserved with formalin will be delayed and it causes environmental pollution. The toxicity of formalin can be reduced by neutralizing the formalin. This study aimed to compare the decrease of mice mass that were preserved with formalin then neutralized with sodium bicarbonate and those that were not neutralized. This study used 18 mice (Mus musculus) which were divided into 3 groups. They were the control group (not given preservative), group preserved with 4% formalin, and group preserved with 4% formalin then neutralized with sodium bicarbonate. All groups of mice were buried for 6 weeks. The changes in mass were assessed with an analysis of the percentage loss in mass. Based on the results of this study, the formalin group had a greater percentage of total mass reduction than the neutralize group. The formalin group had a higher decomposition rate than the neutralizing sodium bicarbonate group. The effectiveness of reducing the concentration of formalin is similar with neutralize group. Therefore, it can be concluded that 4% formalin is recommended for use to accelerate the occurrence of decay and decrease in mass.
... doi: bioRxiv preprint of tamoxifen treatment (Fig 1D). When Immt D/D mice approached a humane endpoint (based on visual assessment of hunched appearance, minimal movement, and bloated abdomen) (19), they presented with enlarged stomachs and fluid retention in their intestines, cecum, and colon ( Fig EV1A). Based on this observation, we first focused our analysis on the intestinal tracts of these animals. ...
The mitochondrial contact site and cristae organizing system (MICOS) is important for cristae junctions (CJ) formation and for maintaining inner mitochondrial membrane (IMM) architecture. As the largest member, MIC60 is the primary scaffold protein for this complex. While MIC60 has been well studied in yeast and cell culture models, its function in mammals is poorly understood. To address this, we developed a mouse model conditionally deleting Immt (which encodes MIC60) and found that global Immt deletion disrupted the MICOS complex and resulted in lethality within 9 days of tamoxifen treatment. Pathologically, these mice display intestinal defects consistent with paralytic ileus, resulting in dehydration. We also identified bone marrow hypocellularity in tamoxifen-treated mice. However, bone marrow transplants from ImmtWT mice failed to rescue survival. Altogether, this novel mouse model demonstrates the importance of MIC60 in vivo, in both hematopoietic and non-hematopoietic tissues, and provides a valuable resource for future mechanistic investigations into the MICOS complex. Such investigations could include an in vivo structure-function analysis of MIC60 functional domains, with characterizations that are relevant to human diseases.
... While changes in body mass give an indication of the animal's health status, conditions such as tumours or ascites may falsely increase body mass therefore body condition scores and humane endpoints for ageing animals were agreed with University Veterinarians at the onset of the study 41,42 . Body condition was scored between 1 to 5 with 3 being the optimal condition, 1 emaciated and 5 obese 42 . ...
Calorie restriction (CR) typically promotes a reduction in body mass which correlates with increased lifespan. We evaluated the overall changes in survival, body mass dynamics, and body composition following long-term graded CR (580 days/19 months) in male C57BL/6J mice. Control mice (0% restriction) were fed ad libitum in the dark phase only (12AL). CR groups were restricted by 10-40% of their baseline food intake (10CR, 20CR, 30CR and 40CR). Body mass was recorded daily, and body composition measured at 8 timepoints. At 728 days/24 months all surviving mice were culled. A gradation in survival rate over the CR groups was found. The pattern of body mass loss differed over the graded CR groups. Whereas the lower CR groups rapidly resumed an energy balance with no significant loss of fat or fat free mass, changes in the 30 and 40CR groups were attributed to higher fat free mass loss and a protection of fat mass. Day to day changes in body mass were less variable under CR than for the 12AL group. There was no indication body mass was influenced by external factors. Partial autocorrelation analysis examined the relationship between daily changes in body masses. A negative correlation between mass on day 0 and mass on day +1 declined with age in the 12AL but not the CR groups. A reduction in the correlation with age suggested body mass homeostasis is a marker of ageing that declines at the end of life and is protected by CR.
... In experiments with water or food restriction, special care has to be taken and, as soon as animals show signs of distress, the restriction protocol is bound to end. Health of animals can be measured in different ways: weight, skin appearance, etc 36 . ...
Fear conditioning is one of the most widely used paradigms in neuroscience to study mechanisms underlying learning and memory and to better understand post-traumatic stress disorders and phobias. Fear conditioning is traditionally achieved by pairing a conditioned stimulus, such as a cue or a context, with an unconditioned stimulus, which is usually a mild foot shock. There are different temporal ways to pair those stimuli generating different contingencies. Learning can occur in one or several sessions, followed by testing sessions in which memory can be assessed at different time points. These testing sessions can also elicit distinct brain mechanisms that take control of the behavioral output, such as reconsolidation or extinction. This implies that protocols which evaluate the course of the memory subsequent to the training session may vary in number. Moreover, different physiological responses can be assessed to measure fear, albeit the most used way by far is to analyze freezing behavior, which involves absence of movement except those related to breathing. In fact, different labs and companies share or sell distinct scripts or softwares for measuring freezing, some of them even involve machine learning. Despite the common knowledge on how to perform experiments using the fear conditioning paradigm, the way in which results are analyzed vary tremendously among papers. This results in lack of reproducibility and comparability between labs, which, altogether, highlights the need of a statistical guideline. This protocol offers a detailed pipeline to perform an array of statistical analyses on freezing behavior in a context of fixed and random, crossed and nested effects, covering classic experimental situations such as repeated behavioral sessions and more than one animal cohort. Although this guide has been designed for fear conditioning experiments, it could be useful to analyze data arising from other behavioral experiments. Analyzing correlated data using tools such as mixed effects models, which account for natural dependence and correlation, can contribute to more reliable and reproducible findings in basic neuroscience research.
... All experimental procedures were conducted within the light period of the light/dark cycle. Even though no adverse effects were expected in this experimental procedure, mice were monitored and humanely euthanized whenever a clinical sign of a humane endpoint was observed 38 The M. avium 2447 inoculum was prepared as previously described 40 . Briefly, a colony of bacteria, from previously infected animals, was harvested and grown until mid-log phase in Middlebrook 7H9 liquid medium with 0.04% Tween 80 at 37 °C. ...
One of the most remarkable findings in the immunology and neuroscience fields was the discovery of the bidirectional interaction between the immune and the central nervous systems. This interplay is tightly regulated to maintain homeostasis in physiological conditions. Disruption in this interplay has been suggested to be associated with several neuropsychiatric disorders. Most studies addressing the impact of an immune system disruption on behavioral alterations focus on acute pro-inflammatory responses. However, chronic infections are highly prevalent and associated with an altered cytokine milieu that persists over time. Studies addressing the potential effect of mycobacterial infections on mood behavior originated discordant results and this relationship needs to be further addressed. To increase our understanding on the effect of chronic infections on the central nervous system, we evaluated the role of Mycobacterium avium infection. A model of peripheral chronic infection with M. avium in female from three mouse strains (Balb/c, C57BL/6, and CD-1) was used. The effect of the infection was evaluated in the cytokine expression profile (spleen and hippocampus), hippocampal cell proliferation, neuronal plasticity, serum corticosterone production and mood behavior. The results show that M. avium peripheral chronic infection induces alterations not just in the peripheral immune system but also in the central nervous system, namely in the hippocampus. Interestingly, the cytokine expression profile alterations vary between mouse strains, and are not accompanied by hippocampal cell proliferation or neuronal plasticity changes. Accordingly, no differences were observed in locomotor, anxious and depressive-like behaviors, in any of the mouse strains used. We conclude that the M. avium 2447 infection-induced alterations in the cytokine expression profile, both in the periphery and the hippocampus, are insufficient to alter hippocampal plasticity and behavior.
... A thin string with one end fastened to the rodent's tail and the other to the top of the cage is used to suspend each mouse in its own cage for the HLU group of mice [10]. Animals are monitored strictly on a daily basis for body weights, food and water intake, physical activity and other signs of distress as described in detail elsewhere [11]. The experimental group is suspended for 21 days (the period was chosen based on prior studies since it demonstrates Future Sci. ...
... The only mice excluded from the present study were those derived from the Pkd1 fl/fl ;Pkhd1-Cre cohorts sacrificed at p14 that were below the 3rd percentile of body weight. These animals were excluded in order to ensure that naturally occurring developmentally delayed runt pups 71 would not bias the present analyses. There is no correlation between the occurrence of runts and the Pkd1 fl/fl ;Pkhd1-Cre genotype [22][23][24]41 . ...
Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent potentially lethal monogenic disorder. Mutations in the PKD1 gene, which encodes polycystin-1 (PC1), account for approximately 78% of cases. PC1 is a large 462-kDa protein that undergoes cleavage in its N and C-terminal domains. C-terminal cleavage produces fragments that translocate to mitochondria. We show that transgenic expression of a protein corresponding to the final 200 amino acid (aa) residues of PC1 in two Pkd1-KO orthologous murine models of ADPKD suppresses cystic phenotype and preserves renal function. This suppression depends upon an interaction between the C-terminal tail of PC1 and the mitochondrial enzyme Nicotinamide Nucleotide Transhydrogenase (NNT). This interaction modulates tubular/cyst cell proliferation, the metabolic profile, mitochondrial function, and the redox state. Together, these results suggest that a short fragment of PC1 is sufficient to suppress cystic phenotype and open the door to the exploration of gene therapy strategies for ADPKD.
... Treated mice were observed for general health and side effects in accordance with standard guidelines. 31 ...
The role of prostaglandins (PGs) in the ovulatory process is known. However, the role of the ATP binding cassette subfamily C member 4 (ABCC4), transmembrane PG carrier protein, in ovulation remains unknown. We report herein that ABCC4 expression is significantly upregulated in preovulatory human granulosa cells (GCs). We found that PGE2 efflux in cultured human GCs is mediated by ABCC4 thus regulating its extracellular concentration. The ABCC4 inhibitor probenecid demonstrated effective blocking of ovulation and affects key ovulatory genes in female mice in vivo. We postulate that the reduction in PGE2 efflux caused by the inhibition of ABCC4 activity in GCs decreases the extracellular concentration of PGE2 and its ovulatory effect. Treatment of female mice with low dose of probenecid as well as with the PTGS inhibitor indomethacin or Meloxicam synergistically blocks ovulation. These results support the hypothesis that ABCC4 has an important role in ovulation and might be a potential target for non-hormonal contraception, especially in combination with PGE2 synthesis inhibitors. These findings may fill the gap in understanding the role of ABCC4 in PGE2 signaling, enhance the understanding of ovulatory disorders, and facilitate the treatment and control of fertility.
... Once a week, the animals were evaluated by two investigators using blind testing-investigators were not aware of the group differences. The general health of the laboratory animals was evaluated according to Burkholder et al. [34]. ...
Adding potassium nitrate (KNO3) to the diet improves the physiological properties of mammalian muscles (rebuilds weakened muscle, improves structure and functionality). The aim of this study was to investigate the effect of KNO3 supplementation in a mouse model. BALB/c mice were fed a KNO3 diet for three weeks, followed by a normal diet without nitrates. After the feeding period, the Extensor digitorum longus (EDL) muscle was evaluated ex vivo for contraction force and fatigue. To evaluate the possible pathological changes, the histology of EDL tissues was performed in control and KNO3-fed groups after 21 days. The histological analysis showed an absence of negative effects in EDL muscles. We also analyzed 15 biochemical blood parameters. After 21 days of KNO3 supplementation, the EDL mass was, on average, 13% larger in the experimental group compared to the controls (p < 0.05). The muscle-specific force increased by 38% in comparison with the control group (p < 0.05). The results indicate that KNO3 has effects in an experimental mouse model, showing nitrate-diet-induced muscle strength. This study contributes to a better understanding of the molecular changes in muscles following nutritional intervention and may help develop strategies and products designated to treat muscle-related issues.
... Automatization allows for monitoring of animals without the presence of an experimenter. Prey animals such as mice and rats may show altered behavior and hide for instance pain, suffering, or distress [96]. Therefore, live cage-side health checks may not detect impaired well-being. ...
Traditionally, in biomedical animal research, laboratory rodents are individually examined in test apparatuses outside their home cages at selected time points. However, the outcome of such tests can be influenced by the novel environment, the time of day, separation from the social group, or the presence of an experimenter. Moreover, valuable information may be missed when the animals are only monitored in short periods. These issues can be overcome by longitudinal monitoring mice and rats in their home cages. To shed light on the development of home cage monitoring (HCM) and the current state of the art, a systematic review was carried out on 521 publications retrieved through PubMed and Web of Science. Both the absolute (~ x26) and relative (~ x7) number of HCM-related publications increased from 1974 to 2020. In both mice and rats, there was a clear bias towards males and individually housed animals, but during the past decade (2011-2020), an increasing number of studies used both sexes and group housing. More than 70% of the studies did not involve a disease model, but the percentage of studies using disease models increased since the 2000s. In most studies, animals were kept for short (up to 4 weeks) length periods in the HCM systems; intermediate length periods (4-12 weeks) increased in frequency in the years between 2011 and 2020. Before the 2000s, HCM techniques were predominantly applied for less than 12 hours, while 24-hour measurements have been more frequently since the 2000s. The systematic review demonstrated that manual monitoring is decreasing but still relevant. Until (and including) the 1990s, most techniques were applied manually but have been progressively replaced by automation since the 2000s. Independent of the publication year, the main behavioral parameters measured were locomotor activity, feeding, and social behaviors; the main physiological parameters were heart rate and electrocardiography. External appearance-related parameters were rarely examined in the home cages. Due to technological progress and application of artificial intelligence, more refined and detailed behavioral parameters could be investigated in the home cage in recent times. Over the period covered in this study, techniques for HCM of mice and rats has improved considerably. This development is ongoing and further progress and validation of HCM systems will extend the applications to allow for continuous, longitudinal, non-invasive monitoring of an increasing range of parameters in group-housed small rodents in their home cages.
... Once a month, the animals were evaluated by two investigators using blind testing, with investigators not knowing differences between groups. The general health of the animals was evaluated according to Burkholder et al. [25] and an assessment table was created (Table 1). The appetite is bad, no evident interest in food or water. ...
Many short-term studies with dietary nitrate supplementation in humans and animal models reported positive effects on the cardiovascular system, exercise efficiency, and immune function. However, there has been long-standing concern related to cancer and adverse hormonal effects. We studied the long-term effects of different potassium nitrate (KNO3) concentrations on laboratory mice longevity and structural changes in their organs. Four groups of male mice were treated with 0 mg (0%), 45 mg (1%), 90 mg (2%), and 140 mg (3%) KNO3 in the drinking water. The groups were monitored for agility and health status daily. The lifespan of mice and organ pathological changes were analyzed. We found no detrimental effects of life-long supplementation of KNO3 on the survival of mice in treatment groups. Nitrate supplementation was associated with a lower level of pathological changes (p = 0.002). We conclude that KNO3 supplementation had no carcinogenic effect on mice and possibly prevented the organs from aging.
... In an optimal condition generally mouse score has been considered as 3, where the bones are palpable but not prominent. Body condition of every mouse was assessed after 35 days treatment and results were demonstrated as Body Condition score (BC score) [37]. ...
Rho-associated coiled-coil kinase (ROCK) inhibition decreases tumourogenic growth, proliferation and angiogenesis. Multifaceted evidences are there about the role of ROCK in cancer progression, but isoform specific analysis in secondary pulmonary melanoma is still unaddressed. This study explored the operating function of ROCK in the metastasis of B16F10 mice melanoma cell line. Inhibition by KD-025 indicated dual wielding role of ROCKII as it is associated with the regulation of MMP9 activity responsible for extra-cellular matrix (ECM) degradation as well as angiogenic invasion as an effect of Src-FAK-STAT3 interaction dependent VEGF switching. We found the assisting role of ROCKII, not ROCKI in nuclear localization of Smads that effectively increased MMP9 expression and activity (p < 0.01). This cleaved the protein components of ECM thereby played a crucial role in tissue remodeling at secondary site during establishment of metastatic tumour. ROCKII phosphorylation at Ser1366 as an activation of the same was imprinted essential for oncogenic molecular bagatelle leading to histo-architectural change of pulmonary tissue with extracellular matrix degradation as a consequence of invasion. Direct correlation of pROCKIISer1366 with MMP9 as well as VEGF expression in vivo studies cue to demonstrate the importance of pROCKIISer1366 inhibition in the context of angiogenesis, and metastasis suggesting ROCKII signaling as a possible target for the treatment of secondary lung cancer specially in metastatic melanoma.
... The hypothesis of this study stated that the suppression of the immune system increased the probability of lung disorders due to respiratory infections, fibrosis, or early malignancy in the lungs when rats were injected with DEN. 34 This is because one rat was not administered DEN died due to lung hemorrhage. This can be explained by Kun MW et al., who discovered the other cause of Wistar rat's lung problem was lung infection due to A.Cantonensis. ...
Background:
Sorafenib is a standard drug used for advanced hepatocellular carcinoma but is often resistant and toxic. Its combination with epigallo-3-catechin gallate leads to reduced resistance and toxicity but an equally effective anti-angiogenic effect.Therefore, this study aims to assess the anti-angiogenic effect of standard-dose Sorafenib compared to the combination of low-dose Sorafenib and epigallo-3-catechin gallate.
Methods:
We conducted an animal study and double-blind, randomized controlled trials. A total of 25 male Wistar rats (7-weeks-old) were randomly divided into four groups, namely Sham (K), Control (O), a combination of low-dose Sorafenib and epigallo-3-catechin gallate group (X1), and standard-dose Sorafenib group (X2). All groups were injected with N-Nitrosodiethylamine 70 mg/kg body weight (BW) intraperitoneally for ten weeks, except the Sham group. After the development of hepatocellular carcinoma, X1 and X2 were treated for two weeks. Subsequently, liver tissues were examined for vascular endothelial growth factor (VEGF) level and microvascular density expression.
Results:
There was a significant difference (p=0.007) in the level of VEGF between group X1 (low dose Sorafenib + EGCG) and X2 (Standard dose Sorafenib). However, the differences in VEGF levels of group X1 and X2 compared to group O(Control) were significantly lower, with values p=0.000136 and p=0.019, respectively. The expression of microvascular density between groups X1 and X2 was not entirely different. Meanwhile, a significant difference (p<0.05) was discovered when both groups were compared with the control group.
Conclusion:
The combination of low-dose Sorafenib with epigallo-3-catechin gallate is superior in reducing the level of VEGF compared to standard-dose Sorafenib and is better than the control. Standard-dose Sorafenib and the combination of low-dose Sorafenib and epigallo-3-catechin gallate have similar effectivity in reducing the expression of microvascular density and could prevent resistance and lower toxicity effects.
... In laboratory animals, body condition evaluations are used as sensitive measures of animal welfare/their health statuses [1][2][3]; moreover, they are easy and non-invasive to obtain. The visual assessment of an animal (according to a standardized scoring system that takes into account fat reserves) is important, because body weight alone may be misleading in the case of, e.g., ascites, tumor growth, or organomegaly [3]. ...
Morphometric data that provide information on body conditions can be used to monitor the health and well-being of animals. In laboratory animals, they can help to evaluate the stress due to experiments or treatments, following the 3R principles. The aim of the present study was to obtain morphometric data of male and female African clawed frogs, Xenopus laevis, as the bases for body condition evaluations. Adult frogs (n = 198) were weighed and standardized photographs were taken. The photographs were used to determine several measurements (length, cranial width, caudal width, thigh width). In addition, a triangle was drawn to outline each frog’s simplified body form, and the triangle surface was calculated. In conclusion, the triangle surface drawn on the dorsal plane of each frog correlated with the body weight of the females. There were significant differences between the body weights and sizes of male and female frogs, with males being smaller (p < 0.001). Based on the morphometric data, females could be assigned to five groups in which an assessment of the animal’s well-being is feasible.
... The closest associated behavioral test for this would be the grimace scale used to measure characteristic facial expressions associated with spontaneous pain in rodents and other mammals [58]. However, the scales have not been widely adopted since they are time-consuming and may be challenging for inexperienced experimenters due to more subjective classifications [59][60][61]. The observation of spontaneous NP was performed only in one of the included studies, by recording the number of freezing episodes when animals had random activities in an open field [19]. ...
Physical activity-based rehabilitative interventions represent the main treatment concept for people suffering from spinal cord injury (SCI). The role such interventions play in the relief of neuropathic pain (NP) states is emerging, along with underlying mechanisms resulting in SCI-induced NP (SCI-NP). Animal models have been used to investigate the benefits of activity-based interventions (ABI), such as treadmill training, wheel running, walking, swimming, and bipedal standing. These activity-based paradigms have been shown to modulate inflammatory-related alterations as well as induce functional and structural changes in the spinal cord gray matter circuitry correlated with pain behaviors. Thus far, the research available provides an incomplete picture of the cellular and molecular pathways involved in this beneficial effect. Continued research is essential for understanding how such interventions benefit SCI patients suffering from NP and allow the development of individualized rehabilitative therapies. This article reviews preclinical studies on this specific topic, goes over mechanisms involved in SCI-NP in relation to ABI, and then discusses the effectiveness of different activity-based paradigms as they relate to different forms, intensity, initiation times, and duration of ABI. This article also summarizes the mechanisms of respective interventions to ameliorate NP after SCI and provides suggestions for future research directions.
... Accordingly, the morphology of liver macrophages and brain microglia did not reveal any signs of inflammation in AAV8-injected mice ( Figure 2H). Moreover, neither body weight nor open-field ambulatory behavior were impacted by AAV8-P3-Alb-mNG or AAV8-P3-IgKL-mNG 4 weeks post-administration ( Figure S1) (Burkholder et al., 2012). Taken together, these experiments demonstrate the minimal footprint of our plasma labeling approach on host physiology and behavior. ...
Studying blood microcirculation is vital for gaining insights into vascular diseases. Blood flow imaging in deep tissue is currently achieved by acute administration of fluorescent dyes in the blood plasma. This is an invasive process, and the plasma fluorescence decreases within an hour of administration. Here, we report an approach for the longitudinal study of vasculature. Using a single intraperitoneal or intravenous administration of viral vectors, we express fluorescent secretory albumin-fusion proteins in the liver to chronically label the blood circulation in mice. This approach allows for longitudinal observation of circulation from 2 weeks to over 4 months after vector administration. We demonstrate the chronic assessment of vascular functions including functional hyperemia and vascular plasticity in micro- and mesoscopic scales. This genetic plasma labeling approach represents a versatile and cost-effective method for the chronic investigation of vasculature functions across the body in health and disease animal models.
... At the time of inoculation, mice were anesthetized using 80 mg/kg body weight ketamine and 10 mg/kg body weight xylazine and infected via intratracheal delivery with the cultured Neisseria subflava (isolate SG-KL01) at 2 x 10 7 CFU or Pseudomonas aeruginosa (strain PAO1) at 1 x 10 6 CFU in 40 mL of sterile phosphate-buffered saline (PBS) once. Mice were monitored for body weight and clinical score (Burkholder et al., 2012). Phenotypic scoring was performed based on indicators of general well-being such as coat condition, normal posture, eyes, and body stance. ...
Neisseria species are frequently identified in the bronchiectasis microbiome, but they are regarded as respiratory commensals. Using a combination of human cohorts, next-generation sequencing, systems biology, and animal models, we show that bronchiectasis bacteriomes defined by the presence of Neisseria spp. associate with poor clinical outcomes, including exacerbations. Neisseria subflava cultivated from bronchiectasis patients promotes the loss of epithelial integrity and inflammation in primary epithelial cells. In vivo animal models of Neisseria subflava infection and metabolipidome analysis highlight immunoinflammatory functional gene clusters and provide evidence for pulmonary inflammation. The murine metabolipidomic data were validated with human Neisseria-dominant bronchiectasis samples and compared with disease in which Pseudomonas-, an established bronchiectasis pathogen, is dominant. Metagenomic surveillance of Neisseria across various respiratory disorders reveals broader importance, and the assessment of the home environment in bronchiectasis implies potential environmental sources of exposure. Thus, we identify Neisseria species as pathobionts in bronchiectasis, allowing for improved risk stratification in this high-risk group.
... Lipocalin-2 and the host defense against Acinetobacter infection were challenged systemically with this infectious dose and monitored for disease severity over the course of infection. Consistent with the observation that the Lcn2-deficient mice exhibited more severe symptoms of infection when employing the lower infectious dose, we found that 60 percent of these mice met humane endpoint criteria by 30 h [119], whilst none of the WT mice required euthanasia at this timepoint (Fig 7). No additional mice met endpoint criteria in the monitoring period after 30 h. ...
Acinetobacter baumannii is an opportunistic pathogen and an emerging global health threat. Within healthcare settings, major presentations of A . baumannii include bloodstream infections and ventilator-associated pneumonia. The increased prevalence of ventilated patients during the COVID-19 pandemic has led to a rise in secondary bacterial pneumonia caused by multidrug resistant (MDR) A . baumannii . Additionally, due to its MDR status and the lack of antimicrobial drugs in the development pipeline, the World Health Organization has designated carbapenem-resistant A . baumannii to be its priority critical pathogen for the development of novel therapeutics. To better inform the design of new treatment options, a comprehensive understanding of how the host contains A . baumannii infection is required. Here, we investigate the innate immune response to A . baumannii by assessing the impact of infection on host gene expression using NanoString technology. The transcriptional profile observed in the A . baumannii infected host is characteristic of Gram-negative bacteremia and reveals expression patterns consistent with the induction of nutritional immunity, a process by which the host exploits the availability of essential nutrient metals to curtail bacterial proliferation. The gene encoding for lipocalin-2 ( Lcn2 ), a siderophore sequestering protein, was the most highly upregulated during A . baumannii bacteremia, of the targets assessed, and corresponds to robust LCN2 expression in tissues. Lcn2 -/- mice exhibited distinct organ-specific gene expression changes including increased transcription of genes involved in metal sequestration, such as S100A8 and S100A9 , suggesting a potential compensatory mechanism to perturbed metal homeostasis. In vitro , LCN2 inhibits the iron-dependent growth of A . baumannii and induces iron-regulated gene expression. To elucidate the role of LCN2 in infection, WT and Lcn2 -/- mice were infected with A . baumannii using both bacteremia and pneumonia models. LCN2 was not required to control bacterial growth during bacteremia but was protective against mortality. In contrast, during pneumonia Lcn2 -/- mice had increased bacterial burdens in all organs evaluated, suggesting that LCN2 plays an important role in inhibiting the survival and dissemination of A . baumannii . The control of A . baumannii infection by LCN2 is likely multifactorial, and our results suggest that impairment of iron acquisition by the pathogen is a contributing factor. Modulation of LCN2 expression or modifying the structure of LCN2 to expand upon its ability to sequester siderophores may thus represent feasible avenues for therapeutic development against this pathogen.
... Hydration was determined by pinching the upper back skin and measuring its relaxation time. 20 Body condition and weight. Each animal was observed visually and palpated on its back and flanks to feel the prominence of its bones. ...
Severe traumatic brain injury (TBI) is a multifactorial injury process involving respiratory, cardiovascular and immune functions in addition to the brain. Thus, live animal models are needed to study the molecular, cellular and systemic mechanisms of TBI. The ethical use of laboratory animals requires that the benefits of approaches be carefully weighed against potential harm to animals. Welfare assessments adapted to severe TBI research are lacking. Here, we introduce a scoresheet to describe and monitor potential distress in animals, which includes general welfare (body weight, general appearance and spontaneous behaviour) and TBI-specific indices (respiratory function, pain, locomotor impairment, wound healing). Implementation of this scoresheet in Sprague–Dawley rats subjected to severe lateral fluid percussion TBI revealed a period of suffering limited to four days, followed by a recovery to normal welfare scores within 10–15 days, with females showing a worse impact than males. The scores indicate that animal suffering in this model is transitory compared with TBI consequences in humans. The scoresheet allows for the implementation of refinement measures including (1) analgesia during the initial period following TBI and (2) humane endpoints set (30% weight loss, score ≥90 and/or respiratory problems). This animal scoresheet tailored to TBI research provides a basis for further refinement of animal research paradigms aimed at understanding or treating the sequelae of severe TBI.
... During the 14-day and 90-day study period, it was observed that the intake of food and water was normal with non-significant body weight variations in both the control and treated groups. Weekly body weight is one of the criteria used to assess the health condition of a tested animal (Tanya et al., 2013). It suggests a pharmacological intervention of lipids, carbohydrates and protein metabolism within the body of the animal because these nutrients play a major role in demonstrating differences in the body's physiological functions. ...
... At 4 days, Sham mice spent more time ambulating than SAH mice (Sidak's post-hoc: P = 0.0454) while no difference in ambulation was observed at 30 days between both groups. Ambulation can be used to assess general health [61,62] and showed that SAH animals are more affected post-surgery. Locomotion was assessed using total distance traveled and velocity. ...
... At 4 days, Sham mice spent more time ambulating than SAH mice (Sidak's post-hoc: P = 0.0454) while no difference in ambulation was observed at 30 days between both groups. Ambulation can be used to assess general health [61,62] and showed that SAH animals are more affected post-surgery. Locomotion was assessed using total distance traveled and velocity. ...
... We observed slightly higher occurrence of sudden death in treated mice compared to control, which was not statistically significant. Other health conditions such as ataxia or dermatitis that were observed in CSF1R-treated mice were all isolated events and considered a common health condition well known to happen spontaneously in laboratory animals (51). Nevertheless, further studies are required to assess the safety and harm-benefit profile of CSF1R inhibitors in long-term treatment of human DMD patients. ...
The role of tissue-resident macrophages during tissue regeneration or fibrosis is not well understood, mainly due to the lack of a specific marker for their identification. Here, we identified three populations of skeletal muscle-resident myelomonocytic cells: a population of macrophages positive for lymphatic vessel endothelial receptor 1 (LYVE1) and T cell membrane protein 4 (TIM4 or TIMD4), a population of LYVE1-TIM4- macrophages, and a population of cells likely representing dendritic cells that were positive for CD11C and major histocompatibility complex class II (MHCII). Using a combination of parabiosis and lineage-tracing experiments, we found that, at steady state, TIM4- macrophages were replenished from the blood, whereas TIM4+ macrophages locally self-renewed [self-renewing resident macrophages (SRRMs)]. We further showed that Timd4 could be reliably used to distinguish SRRMs from damage-induced infiltrating macrophages. Using a colony-stimulating factor 1 receptor (CSF1R) inhibition/withdrawal approach to specifically deplete SRRMs, we found that SRRMs provided a nonredundant function in clearing damage-induced apoptotic cells early after extensive acute injury. In contrast, in chronic mild injury as seen in a mouse model of Duchenne muscular dystrophy, depletion of both TIM4-- and TIM4+-resident macrophage populations through long-term CSF1R inhibition changed muscle fiber composition from damage-sensitive glycolytic fibers toward damage-resistant glycolytic-oxidative fibers, thereby protecting muscle against contraction-induced injury both ex vivo and in vivo. This work reveals a previously unidentified role for resident macrophages in modulating tissue metabolism and may have therapeutic potential given the ongoing clinical testing of CSF1R inhibitors.
... We did, however, observe a severe, progressive weight loss throughout the duration of the experiment in all mice. We believe that the rapid change in weight is primarily due to a stop in water intake, 29,57,58 which is consistent with the fact that a similar weight loss is observed in studies in which mice have a restricted access to water in order to motivate behavioural training. 59 Dehydration may have influenced the water content of the blood and, therefore, been a contributor to the changes in the flow dynamics of the cerebral vasculature observed here during acute inflammation. ...
Systemic inflammation affects cognitive functions and increases the risk of dementia. This phenomenon is thought to be mediated in part by cytokines that promote neuronal survival, but the continuous exposure to which may lead to neurodegeneration. The effects of systemic inflammation on cerebral blood vessels, and their provision of adequate oxygen to support critical brain parenchymal cell functions, remains unclear. Here, we demonstrate that neurovascular coupling is profoundly disturbed in lipopolysaccharide (LPS) induced systemic inflammation in awake mice. In the 24 hours following LPS injection, the hyperaemic response of pial vessels to functional activation was attenuated and delayed. Concurrently, under steadystate conditions, the capillary network displayed a significant increase in the number of capillaries with blocked blood flow, as well as increased duration of ‘capillary stalls’—a phenomenon previously reported in animal models of stroke and Alzheimer’s disease pathology. We speculate that vascular changes and impaired oxygen availability may affect brain functions following acute systemic inflammation and contribute to the long-term risk of neurodegenerative changes associated with chronic, systemic inflammation.
... The monitoring of physical activity is a widespread tool to evaluate physical health in humans and animals alike. Physical activity is a complex multi-dimensional behavior that is characterized by frequency, duration, intensity and mode [1,2]. Typical metrics to quantify activity are locomotion (e.g., steps and traveled distance) and heart rate. ...
In this work, a novel method is presented for non-contact non-invasive physical activity monitoring, which utilizes a multi-axial inertial measurement unit (IMU) to measure activity-induced structural vibrations in multiple axes. The method is demonstrated in monitoring the activity of a mouse in a husbandry cage, where activity is classified as resting, stationary activity and locomotion. In this setup, the IMU is mounted in the center of the underside of the cage floor where vibrations are measured as accelerations and angular rates in the X-, Y- and Z-axis. The ground truth of activity is provided by a camera mounted in the cage lid. This setup is used to record 27.67 h of IMU data and ground truth activity labels. A classification model is trained with 16.17 h of data which amounts to 3880 data points. Each data point contains eleven features, calculated from the X-, Y- and Z-axis accelerometer data. The method achieves over 90% accuracy in classifying activity versus non-activity. Activity is monitored continuously over more than a day and clearly depicts the nocturnal behavior of the inhabitant. The impact of this work is a powerful method to assess activity which enables automatic health evaluation and optimization of workflows for improved animal wellbeing.
... Ultimately, we used 7 mice per group; fortunately, we had no losses and did not have to use extra mice for our experiments. Mice were monitored for behavioral and physical signs of animal welfare [27] and observed for at least 1 h each day by two investigators blinded to treatments, and the mice remained within acceptably humane endpoint criteria with no indication of pain and distress outside of the experimental stimulus and obesity because of treatments. At the end of the experiments, all mice were humanely euthanized using high-dose anesthesia (pentobarbital, 100 mg/kg, i.p.) followed by cervical dislocation. ...
Background
An association between dysregulated glucose levels in patients with diabetes mellitus and detrimental effects on the central nervous system, particularly in Alzheimer disease, has been recognized. Atorvastatin treatment has improved memory and cognition in some patients with diabetes mellitus and Alzheimer disease.
Objectives
To determine possible neuroprotective effects of atorvastatin on memory and cognition by measuring changes in an adverse stimulus avoidance learning deficit induced by alloxan in a murine model of diabetes mellitus and impaired memory and cognition.
Methods
We administered 150 mg/kg and 100 mg/kg alloxan in saline (intraperitoneally, i.p.) at a 48 h interval to produce a model of diabetes mellitus in male BALB/c mice. An oral glucose tolerance test (OGTT) was used to assess blood glucose regulation. After demonstrating hyperglycemia in mice (n = 7 per group) we administered vehicle (saline, i.p.), atorvastatin (10 mg/kg, i.p.), or liraglutide (200 μg/kg, i.p.) for 28 d except for those in a negative control group, which were given saline instead of alloxan, and a group administered atorvastatin alone, which were given saline instead of alloxan followed by atorvastatin (10 mg/kg, i.p.) for 28 d. Locomotor activity was measured 24 h after the final drug treatments, and subsequently their learned behavioral response to an adverse electrical stimulus to their plantar paw surface in a dark compartment was measured using a passive avoidance apparatus (Ugo Basile) in a model of impaired memory and cognition associated with Alzheimer disease. To determine any deficit in their learned avoidance of the adverse stimulus, we measured the initial latency or time mice spent in an illuminated white compartment before entering the dark compartment in the learning trial, and on the day after learning to avoid the adverse stimulus, the retention period latency in the light compartment and time spent in the dark compartment.
Results
Atorvastatin alone produced no significant change in blood glucose levels ( F 4,10 = 0.80, P = 0.55) within 2 h. Liraglutide decreased blood glucose levels after 0.5 h ( F 4,10 = 11.7, P < 0.001). We found no significant change in locomotor activity in any group. In mice with alloxan-induced diabetes, atorvastatin significantly attenuated the decreased avoidance associated with the diabetes ( F 4,30 = 38.0, P = 0.02) and liraglutide also significantly attenuated the decreased avoidance ( F 4,30 = 38.0, P < 0.001). Atorvastatin alone had no significant effect on the adversive learned response compared with vehicle treatment ( F 4,30 = 38.0, P > 0.05). Atorvastatin significantly decreased the time mice with alloxan-induced diabetes spent in the dark compartment compared with mice in the diabetes group without atorvastatin treatment ( F 4,30 = 53.9, P = 0.046). Liraglutide also significantly reduced the time mice with alloxan-induced diabetes spent in the dark compartment compared with vehicle-treated mice with alloxan-induced diabetes ( F 4,30 = 53.9, P < 0.001). Atorvastatin treatment alone had no significant effect on the time mice spent in dark compartment compared with the control group ( F 4,30 = 53.9, P > 0.05).
Conclusion
Atorvastatin significantly attenuated the adverse stimulus avoidance learning deficit in the alloxan-induced murine model of diabetes suggesting decreased impairment of memory and cognition.
... An indicator of potential carcinogenesis in mice, often before detection of overt tumor formation, is abnormal weight change (51). Thus, all animals were weighed on a regular basis, and the time time-dependent weight changes are shown in Fig. 6. ...
Far‐UVC radiation, typically defined as 200‐235 nm, has similar or greater anti‐microbial efficacy compared to conventional 254‐nm germicidal radiation. In addition, biophysical considerations of the interaction of far‐UVC with tissue, as well as multiple short‐term safety studies in animal models and humans, suggest that far‐UVC exposure may be safe for skin and eye tissue. Nevertheless, the potential for skin cancer after chronic long‐term exposure to far‐UVC has not been studied. Here, we assessed far‐UVC induced carcinogenic skin changes and other pathological dermal abnormalities in 96 SKH‐1 hairless mice of both sexes that were exposed to average daily dorsal skin doses of 400 mJ/cm2, 130 mJ/cm2 or 55 mJ/cm2 of 222 nm far‐UVC radiation for 66 weeks, 5 days per week, 8 hours per day, as well as similarly‐treated unexposed controls. No evidence for increased skin cancer, abnormal skin growths, or incidental skin pathology findings was observed in the far‐UVC exposed mice. In addition, there were no significant changes in morbidity or mortality. The findings from this study support the long‐term safety of long‐term chronic exposure to far‐UVC radiation, and therefore its potential suitability as a practical anti‐microbial approach to reduce airborne viral and bacterial loads in occupied indoor settings.
... pain and distress and 4 indicating moribund condition(44). Surviving mice were checked for parasitemia. ...
While the asexual cycle of Toxoplasma gondii can occur in any warm-blooded animal, the sexual cycle is restricted to the feline intestine. We previously determined that because cats lack delta-6-desaturase activity in their intestines, they build up excess linoleic acid, which signals T. gondii to undergo sexual development. We hypothesized that T. gondii oxygenates linoleic acid to signal sexual development, so we examined the T. gondii genome for potential lipoxygenases (TgLOX) enzymes. We identified seven potential TgLOXs that were at least 100-fold more abundant in the cat intestinal versus the tissue culture tachyzoite stage. Parasites deleted in TgLOX1 (TgΔLOX1) had no significant growth differences in tissue culture fibroblast cells. Because the sexual development assay begins with brain cysts, we infected mice with TgΔLOX1 and were surprised to find that TgΔLOX1 had reduced virulence. The TgΔLOX1 parasitemia was reduced by 3 days postinfection and largely cleared by 7 days postinfection. At 3 days postinfection, the cytokines IFNγ, IL-6, MCP-1, and TNF-α were significantly reduced in TgΔLOX1-infected mice, which prompted us to examine TgΔLOX1 in IFNγKO mice. We found that IFNγKO mice infected with TgΔLOX1 succumbed to acute infection with the same kinetics as the parental and complemented strains, suggesting the role of TgLOX1 in mice was IFNγ dependent. In tissue culture fibroblasts, TgLOX1 was localized within the parasite, but in leukocytes from infected mice and activated macrophages, TgLOX1 was localized in vesicular structures in the host cytoplasm. These results suggest that TgLOX1 in these vesicular structures modifies the host immune response.
Importance
Lipoxygenases are enzymes that catalyze the dioxygenation of polyunsaturated fatty acids such as linoleic and arachidonic acid. These modifications create signaling molecules that are best characterized for modulating the immune response. Deletion of the first lipoxygenase characterized for Toxoplasma gondii (TgLOX1) generated a less virulent strain and infected mice showed a decreased immune response. This virulence defect was dependent on the mouse cytokine IFNγ. TgLOX1 changes location from inside the parasite in tissue culture conditions to vesicular structures within the host immune cells during mouse infection. These results suggest that TgLOX1 plays a role in the modification of the host immune response in mice.
... Mice were housed 5-6 per cage with ad libitum access to standard laboratory chow and water under a 12-h light/dark cycle. According to previously defined criteria [33], mice displaying tumors or physical abnormalities were excluded from the study and euthanized by an overdose of pentobarbital (30 mg, intraperitoneally [i.p.]). All efforts were made to reduce the number of animals used in agreement with the European Communities Council Directive (24 November 1986, 86/609/EEC). ...
Early misfolded superoxide dismutase 1 (mfSOD1) accumulation, motor neuron (MN) degeneration, and microgliosis are hallmark pathological features in SOD1G93A amyotrophic lateral sclerosis (ALS) mice. Because of the different vulnerabilities of distinct MN subtypes, degenerating and surviving MNs coexist in different proportions during disease progression. By examining the expression of misfolded conformers of SOD1 using specific antibodies, we defined distinct MN phenotypes that were evaluated during disease progression and the local neuroinflammatory reaction. The most severe phenotype corresponded to somata of fast-twitch subtype MNs, which exhibited highly positive mfSOD1 immunostaining and an extreme degree of vacuolar degeneration. Vacuoles, which are of mitochondrial origin, contain mfSOD1 in conjunction with nonmitochondrial proteins, such as chromogranin, CD81, and flotillin. The fusion of ER-derived vesicles enriched in mfSOD1 with outer mitochondrial membranes is thought to be the primary mechanism for vacuole formation. In addition, the ulterior coalescence of enlarged mitochondria may lead to the formation of giant vacuoles. Vacuolar degeneration is a transient degenerative process occurring early during the presymptomatic stages of the disease in ALS mice. Some vacuolated MNs are also positive for pMLKL, the effector protein of necroptosis. This indicates a newly described mechanism in which extracellular vesicles derived from damaged MNs, via cellular secretion or necroptotic disruption, may be the triggers for initiating neuroinflammation, glial-mediated neurotoxicity, and disease spreading. Furthermore, as MN degeneration in mutant SOD1 mice is noncell autonomous, the effects of experimentally increasing or decreasing the microglial response on the expression of MN phenotypes were also evaluated, demonstrating bidirectional cross talk signaling between the degree of expression of mfSOD1 and local neuroinflammation. More detailed knowledge regarding these processes occurring long before the end stages of the disease is necessary to identify novel molecular targets for future preclinical testing.
... No alteration in the general health status of stressed breeders emerged at the end of the stress paradigm. The health measures were taken by the animal caretakers through the daily observation of the animals in their home cage in order to assess both behavioral and physical indicators of welfare 39 . These included hunched posture, dull or sluggish movements, reduced locomotion/immobility, altered nest building and stereotypic behaviors, excessive grooming, absence of feces, rough hair coat, squinted eyes, skin abrasions/lesions 39 . ...
Fragile X Syndrome (FXS) is the most common heritable form of mental retardation and monogenic cause of autism spectrum disorder (ASD). FXS is due to a mutation in the X-linked FMR1 gene and is characterized by motor, cognitive and social alterations, mostly overlapping with ASD behavioral phenotypes. The severity of these symptoms and their timing may be exacerbated and/or advanced by environmental adversity interacting with the genetic mutation. We therefore tested the effects of the prenatal exposure to unpredictable chronic stress on the behavioral phenotype of juveniles of both sexes in the Fmr1 knock-out (KO) mouse model of FXS. Mice underwent behavioral tests at 7–8 weeks of age, that is, when most of the relevant behavioral alterations are absent or mild in Fmr1-KOs. Stress induced the early appearance of deficits in spontaneous alternation in KO male mice, without exacerbating the behavioral phenotype of mutant females. In males stress also altered social interaction and communication, but mostly in WT mice, while in females it induced effects on locomotion and communication in mice of both genotypes. Our data therefore highlight the sex-dependent relevance of early environmental stressors to interact with genetic factors to influence the appearance of selected FXS- and ASD-like phenotypes.
Obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist, has been demonstrated to ameliorate the histopathological characteristics of liver damage. Nonetheless, the systemic safety profile of OCA with regard to reproduction and development remains poorly understood. In the present study, we conducted a dose-response experiment by administering OCA at doses of 5 mg/kg, 10 mg/kg, or 20 mg/kg through tube feeding to investigate its effect on reproductive development and fertilization rate in both male and female mice. Furthermore, we evaluated the levels of protein and mitochondrial function in the placenta through western blot, qPCR, and scanning electron microscopy. The results showed that 10 mg/kg and 20 mg/kg OCA doses significantly reduced the rate of placental implantation (P < 0.05). Also, OCA increased maternal body weight. In addition, OCA increased levels of FXR and TGR5 and produced changes in oxidative stress levels (P < 0.05). Mitochondrial activity result found that 10 mg/kg and 20 mg/kg of OCA significantly reduced the mitophagy autosomes/nucleus compared with the normal control group (P < 0.05). What is more, there was no significant difference in sperm count after OCA intervention in either C57BL/10 mice or BALB/c mice. Overall, we demonstrated that OCA treatment protected against placental implantation by suppressing placental oxidative stress and mitochondrial activity.
While the asexual cycle of Toxoplasma gondii can occur in any warm-blooded animal, the sexual cycle is restricted to the feline intestine. We previously determined that because cats lack delta-6-desaturase activity in their intestines, they build up excess linoleic acid, which signals T. gondii to undergo sexual development. We hypothesized that T. gondii oxygenates linoleic acid to signal sexual development, so we examined the T. gondii genome for lipoxygenases-like enzymes (TgLOXL) enzymes. We identified seven TgLOXLs that were at least 100-fold more abundant in the cat intestinal versus the tissue culture tachyzoite stage. Parasites deleted in TgLOXL1 (TgΔLOXL1) had no significant growth differences in tissue culture fibroblast cells. Because the sexual development assay begins with brain cysts, we infected mice with TgΔLOXL1 and were surprised to find that TgΔLOXL1 had reduced virulence. The TgΔLOXL1 parasitemia was reduced by 3 d post-infection and largely cleared by 7 d post-infection. At 3 d post-infection, the cytokines interferon gamma (IFN-γ), IL-6, MCP-1, and TNF-α were significantly reduced in TgΔLOXL1-infected mice, which prompted us to examine TgΔLOXL1 in IFN-γ KO mice. We found that IFN-γ KO mice infected with TgΔLOXL1 succumbed to acute infection with the same kinetics as the parental and complemented strains, suggesting that TgLOXL1 plays a role in the IFN-γ signaling cascade. In tissue culture fibroblasts, TgLOXL1 was localized within the parasite, but in leukocytes from infected mice and activated macrophages, TgLOXL1 was localized within the host cytoplasm. These results suggest that TgLOXL1 changes localization in response to host immune activation.
IMPORTANCE
Lipoxygenases (LOXs) are enzymes that catalyze the deoxygenation of polyunsaturated fatty acids such as linoleic and arachidonic acid. These modifications create signaling molecules that are best characterized for modulating the immune response. Deletion of the first lipoxygenase-like enzyme characterized for Toxoplasma gondii (TgLOXL1) generated a less virulent strain, and infected mice showed a decreased immune response. This virulence defect was dependent on the mouse cytokine interferon gamma IFNγ. TgLOXL1 changes location from inside the parasite in tissue culture conditions to vesicular structures within the host immune cells during mouse infection. These results suggest that TgLOXL1 plays a role in the modification of the host immune response in mice.
The development of effective and flexible vaccine platforms is a major public health challenge, especially in the context of influenza vaccines that have to be renewed every year. Adenoviruses (AdVs) are easy to produce and have a good safety and efficacy profile when administered orally, as demonstrated by the long-term use of oral AdV-4 and -7 vaccines in the U.S. military. These viruses therefore appear to be the ideal backbone for the development of oral replicating vector vaccines. However, research into these vaccines is limited by the ineffectiveness of human AdV replication in laboratory animals. The use of mouse AdV type 1 (MAV-1) in its natural host allows infection to be studied under replicating conditions. Here, we orally vaccinated mice with a MAV-1 vector expressing influenza hemagglutinin (HA) to assess the protection conferred against an intranasal challenge of influenza. We showed that a single oral immunization with this vaccine generates influenza-specific and -neutralizing antibodies and completely protects mice against clinical signs and viral replication, similar to traditional inactivated vaccines.
IMPORTANCE Given the constant threat of pandemics and the need for annual vaccination against influenza and possibly emerging agents such as SARS-CoV-2, new types of vaccines that are easier to administer and therefore more widely accepted are a critical public health need. Here, using a relevant animal model, we have shown that replicative oral AdV vaccine vectors can help make vaccination against major respiratory diseases more available, better accepted, and therefore more effective. These results could be of major importance in the coming years in the fight against seasonal or emerging respiratory diseases such as COVID-19.
The research wants to find out if curcumin in turmeric is effective in treating inflammations of the eye. Theresearch also aims to determine if curcumin is an irritant. To answer these questions, curcumin wasextracted and eyedrops were made to be tested on animal models of eye inflammation. Results show thatsigns of inflammation was alleviated with the use of the eyedrops made from curcumin. The study alsofound out that curcumin is practically non-irritative. The study shows the potential use of curcumin as anophthalmic drop, capable of eliminating eye inflammations.
Effective wound management imposes several challenges in clinical outcomes due to the complexity of the wound microenvironment, bacterial infections, impaired angiogenesis, aggravated inflammation, and enduring pain. In addition, adhesion on wet biological tissue is another extremely challenging task. Addressing all the issues is necessary for an effective wound healing process. Herein, we developed a unique multifunctional, adhesive composite hydrogel composed of gelatin, chitosan, polydopamine-coated bioactive glass (BG), and curcumin-capped silver nanoparticles (Cur-AgNPs) to target the multifaceted complexity of the wound. The PDA-coated BG serves multiple purposes: (1) adhesivity: catechol groups of PDA and Ca ion released from BG chelate the group present in the hydrogel network and surrounding tissues, (2) angiogenesis: promotes vascularization due to the release of Si from BG, and (3) BG also serves as the "reservoir" for the pain-relieving diclofenac sodium drug with a sustained-release behavior. Cur-AgNPs provide excellent bactericidal and anti-inflammatory properties to the composite hydrogel. In situ application of the composite hydrogel could serve the purpose of a "skin biomimetic" and work as a barrier along with bactericidal properties to inhibit the microbial growth. The multifunctional composite hydrogel (MCH) targeted multiple aspects of wound repair including pain alleviation, elimination of microbes (up to 99%), reduced inflammation, high adhesivity, and increased angiogenesis for effective skin regeneration. The MCH showed excellent wound healing potential as significant wound closure was observed at day 7 and also significantly upregulated the expression of crucial genes involved in the skin regeneration process along with increasing vascularization in the wound area.
Biolimus A9 (BA9) is a novel rapamycin derivative. In this report we evaluated the potential toxicity of BA9 in a developmental and reproduction toxicity study (segment Ⅰ, Ⅱ, Ⅲ). In segment I, body weight gains in F0 rats receiving 0.80mg/kg/day were decreased. A lower fertility index of males was observed and females failed to become pregnant in the 0.80g/kg/day group. The number of live fetuses and implantations were decreased while the number of dead fetuses, resorptions, and implantation losses were increased in the 0.12mg/kg/day group. In segment Ⅱ, maternal toxicity: body weight gains in F0 females receiving 0.036 and 0.090mg/kg/day group were decreased. Embryo toxicity: In the 0.090mg/kg group，weights and body lengths of fetuses were decreased, the number of viable fetuses was decreased and resorbed fetuses increased. Teratogenic effects: The percent of visceral variations and skeletal variations were both increased in the 0.090mg/kg/day group. In segment Ⅲ, F0 rats receiving BA9 at doses of 0.12 and 0.8mg/kg resulted in reproductive and maternal toxicity, consisting of prolonged labor, dystocia, increased mortality, along with reductions in lactation food consumption. F1 rats in the 0.12mg/kg/day group showed reproductive and developmental toxicity consisting of body weight decreases, decreased food consumption after weaning and a reduction in the gestation index of pregnant rats. Based on these findings, the no-observed-adverse-effect-level (NOAEL) of BA9 toxicity in segment Ⅰ and Ⅲ was 0.02mg/kg/day. The NOAEL in segment Ⅱ was 0.015mg/kg/day.
The demand of exploring strategies to enhance chemotherapy drug efficacy and alleviate adverse effects by using natural compounds is increasing. Sinensetin (SIN) is a kind of natural flavonoids with anti-inflammatory activities. However, its protective impact on chemotherapy-induced adverse effects has not been well demonstrated. Here, we found that SIN could inhibit Cisplatin-induced release of proinflammatory cellular contents and inflammatory cell death-pyroptosis. In addition, Cisplatin-induced activation of gasdermin E (GSDME), a critical mediator of chemotherapy-induced tissue injury, could also be reversed by SIN. Furthermore, SIN impaired Cisplatin-induced intracellular damages, including ROS release and DNA damages. Importantly, SIN was able to alleviate intestinal injury in Cisplatin-challenged mice, which was accompanied by the decrease of lytic cell death and immune cell infiltration. Of note, SIN administration did not reverse Cisplatin-caused tumor suppression in vivo. In conclusion, our result provides a potential application of SIN to reduce Cisplatin-caused adverse effects, without impairing its anti-tumor capacity.
Background: Sorafenib is a standard drug used for advanced hepatocellular carcinoma but is often resistant and toxic. Its combination with epigallo-3-catechin gallate leads to reduced resistance and toxicity but an equally effective anti-angiogenic effect.Therefore, this study aims to assess the anti-angiogenic effect of standard-dose Sorafenib compared to the combination of low-dose Sorafenib and epigallo-3-catechin gallate. Methods: We conducted an animal study and double-blind, randomized controlled trials. A total of 25 male Wistar rats (7 weeks old) were randomly divided into four groups, namely Sham (K), Control (O), a combination of low-dose Sorafenib and epigallo-3-catechin gallate group (X1), and standard-dose Sorafenib group (X2). All groups were injected with N-Nitrosodiethylamine 70 mg/kg body weight (BW) intraperitoneally for ten weeks, except the Sham group. After the development of hepatocellular carcinoma, X1 and X2 were treated for two weeks. Subsequently, liver tissues and tumor masses were examined for vascular endothelial growth factor (VEGF) level and microvascular density expression. Results: There was a significant difference (p=0.007) in the level of VEGF between group X1 (low dose Sorafenib + EGCG) and X2 (Standard dose Sorafenib). However, the differences in VEGF levels of group X1 and X2 compared to group O(Control) were significantly lower, with values p=0.000136 and p=0.019, respectively. The expression of microvascular density between groups X1 and X2 was not entirely different. Meanwhile, a significant difference (p
Fragile X syndrome (FXS) is a major neurodevelopmental disorder and the most common monogenic cause of autism spectrum disorder (ASD). FXS is caused by a mutation in the X-linked FMR1 gene leading to the absence of the FMRP protein, inducing several behavioral deficits, including motor, emotional, cognitive, and social abnormalities. Beside its clear genetic origins, FXS can be modulated by environmental factors, e.g., stress exposure: indeed the behavioral phenotype of FXS, as well as of ASD patients can be exacerbated by the repeated experience of stressful events, especially early in life. Here we investigated the long-term effects of prenatal exposure to unpredictable chronic stress on the behavioral phenotype of the Fmr1-knock-out (KO) mouse model for FXS and ASD. Mice were tested for FXS- and ASD-relevant behaviors first at adulthood (3 months) and then at aging (18 months), in order to assess the persistence and the potential time-related progression of the stress effects. Stress induced the selective emergence of behavioral deficits in Fmr1-KO mice that were evident in spatial memory only at aging. Stress also exerted several age-specific behavioral effects in mice of both genotypes: at adulthood it enhanced anxiety levels and reduced social interaction, while at aging it enhanced locomotor activity and reduced the complexity of ultrasonic calls. Our findings underline the relevance of gene-environment interactions in mouse models of neurodevelopmental syndromes and highlight the long-term behavioral impact of prenatal stress in laboratory mice.
Probiotics have gained importance in recent years as a viable substitute to antibiotics for boosting livestock performance. Along with enhanced nutrient digestibility and immunomodulation, probiotics have reflected an enormous reduction in gastrointestinal tract infection through in-feed usage. Although, each novel probiotic strain cannot be presumed to share a historical safety perspective with conventional strains. Harmful effects of probiotics can be dependent on prevailing immunological conditions, strain specified, and physiological conditions of the host. The most important consideration is the strain’s stability. Probiotics state an effective chance of replacing antibiotics in animals, their safety measures should have been adhered to for safety concerns. The chapter aims to provide the several tests for evaluating the general health of the animals after probiotic treatment such as, Cylinder, Irwin, Wire suspension, and vertical pole tests. These tests are being utilized for past years to enable practical evaluation of animal species in laboratory by following these protocols. They are scientifically evident and experimentally safe as per the requirements to avoid animal suffering during analysis.Key wordsProbioticsAnimal healthNutrition
Rho-associated coiled-coil kinase (ROCK) inhibition decreases tumourogenic growth, proliferation and angiogenesis. Multifaceted evidences are there about the role of ROCK in cancer progression, but isoform specific analysis in secondary pulmonary melanoma is still unaddressed. This study explored the operating function of ROCK in the metastasis of B16F10 mice melanoma cell line. Inhibition by KD-025 indicated dual wielding role of ROCKII as it is associated with the regulation of MMP9 activity responsible for extra-cellular matrix (ECM) degradation as well as angiogenic invasion as an effect of Src-FAK-STAT3 interaction dependent VEGF switching. We found the assisting role of ROCKII, not ROCKI in nuclear localization of Smads that effectively increased MMP9 expression and activity (p < 0.01). This cleaved the protein components of ECM thereby played a crucial role in tissue remodeling at secondary site during establishment of metastatic tumour. ROCKII phosphorylation at Ser1366 as an activation of the same was imprinted essential for oncogenic molecular bagatelle leading to histo-architectural change of pulmonary tissue with extracellular matrix degradation as a consequence of invasion. Direct correlation of pROCKIISer1366 with MMP9 as well as VEGF expression in vivo studies cue to demonstrate the importance of pROCKIISer1366 inhibition in the context of angiogenesis, and metastasis suggesting ROCKII signaling as a possible target for the treatment of secondary lung cancer specially in metastatic melanoma.
Studying blood microcirculation is vital for gaining insights into vascular diseases. Acute administration of fluorescent tracers is currently used for deep tissue blood flow imaging. This is invasive, and the plasma fluorescence decreases within an hour of administration. We report a novel approach for the longitudinal study of vasculature. Using a single systemic administration of viral vectors, we express fluorescent secretory albumin-fusion proteins in the liver to label the blood in mice. All segments of the vasculature in brain and peripheral tissue are observable by two-photon microscopy within two weeks of vector administration. This approach allows for observation of circulation without the need for repeated administration for several months. We demonstrate the chronic assessment of vascular functions at micro- and mesoscopic scales. This genetic plasma labeling approach represents a versatile and cost-effective method for the chronic investigation of vasculature functions across the body in health and disease.
Transgenic and knockout technologies have caused a shift in research focus at many institutions. Increased numbers of mice—some with potentially debilitating phenotypes—are being maintained to their natural life spans, and this has chal- lenged the laboratory animal community to devise effective strategies for monitoring and managing these colonies. Aside from meeting essential obliga- tions for ensuring the well-being (freedom from discomfort, distress, and pain) of the research mouse, effective management and supervision of mouse colonies is useful in fulfilling the "3 Rs" (replacement, reduc- tion, refinement) put forth by Russell and Burch1. We also suggest a fourth essential "R," investigator responsibility, since inves- tigators are increasingly monitoring and managing their own transgenic colonies. A well-monitored and -managed rodent colony will provide: more precise informa- tion on the progression of disease or phe- notype; clear criteria for data collection; recovery of data potentially lost when unmonitored mice die; improved quality, and increased uniformity, of data due to standardized techniques for monitoring health; additional observations and charac- terization of phenotypes; possible decrease in number of mice needed to characterize a phenotype; and decreased costs of charac- terizing a phenotype.
Murine ulcerative dermatitis (UD) is a common progressive condition of mice with a C57BL/6 background. Typically, mice present with scabs and crusts on the skin of the dorsal neck and ears, and are often severely pruritic. Animals tend to scratch the lesions, causing additional trauma to the already ulcerated and inflamed skin. Therapeutic intervention largely has been unsuccessful, in part due to the lack of a known cause for the disease. Though the exact etiology of UD has not been elucidated, substance P (SP) has recently been demonstrated as an important neuropeptide linked to the itch-scratch cycle. SP functions at the tachykinin neurokinin 1 (NK1) receptor. We hypothesized that inhibition of SP binding to the NK1 receptor would decrease the itch sensation, thus decreasing scratching behavior and subsequent skin trauma. The purpose of this study was to evaluate the effectiveness of an NK1 receptor antagonist, maropitant citrate, as a treatment for murine UD. Treatment with 1 mg/kg maropitant citrate significantly reduced the size of UD lesions in mice.
Facial expression is widely used as a measure of pain in infants; whether nonhuman animals display such pain expressions has never been systematically assessed. We developed the mouse grimace scale (MGS), a standardized behavioral coding system with high accuracy and reliability; assays involving noxious stimuli of moderate duration are accompanied by facial expressions of pain. This measure of spontaneously emitted pain may provide insight into the subjective pain experience of mice.
Accurate, rapid, and noninvasive health assessments are required to establish more appropriate endpoints in mouse cancer models where tumor size is not easily measured. We evaluated potential endpoints in mice with experimentally induced peritoneal lymphoma, an abdominal tumor model, by comparing body weight, body condition, and behavior with those of a control group of mice not developing lymphoma. Our hypothesis was that body weight would increase or plateau, whereas body condition and behavioral scores would decrease, as disease progressed. Results indicated that body weight did not differ significantly between the control and experimental groups, but the experimental group experienced significant decreases in both body condition and behavioral scores. Our results support the use of body condition and behavioral scoring as adjunctive assessment methods for mice involved in abdominal lymphoma tumor studies in which health may decline despite an increase or plateau in body weight.
Environmental enrichment of laboratory mice can improve the quality of research, but debate arises over the means of enrichment and its ability to be used in a sterile environment. One important form of enrichment is nesting material. Mice in the wild build dome-shaped, complex, multilayered nests, but this behavior is not seen in the laboratory, perhaps due to inappropriate nesting material rather than the nest-building ability of the mice. Here we focus on the use of naturalistic nesting materials to test whether they improve nest quality through the use of a 'naturalistic nest score' system; we also focus on materials that can be sterilized and easily used in existing housing systems. We first determined whether C57BL/6J mice build naturalistic nests when given shredded paper strips. We then compared these shredded paper strips with other commonly used nesting enrichments (facial tissues and compressed cotton squares). Nests were scored for 6 d. We found that the shredded paper strips allowed the mice to build higher quality nests than those built with any of the other materials. Nests built with tissues were of intermediate quality, and nests built with compressed cotton squares were of poor quality, similar to those built by the control group. These results suggest that C57BL/6J mice given appropriate nesting materials can build nests similar to those built by their wild counterparts.
This study evaluated whether the automated behaviour recognition software 'HomeCageScan' (HCS) could detect behaviour changes and any positive analgesic effects in two mouse strains undergoing vasectomy (C3H/HeNCrl and C57BL/6). Another objective was to test the effectiveness of HCS in differentiating between the effects of each treatment relative to conventional manual analysis. Each control (unoperated) group consisted of four mice of each strain. They were either untreated mice, mice given meloxicam alone (10 mg/kg) or mice given either saline or meloxicam (10 mg/kg) 30 min prior to isoflurane anaesthesia. The vasectomized mice received either saline or meloxicam at 5, 10 or 20 mg/kg, again, 30 min prior to isoflurane anaesthesia. Filming began one hour following surgery. Each mouse was filmed for 6 min for the manual analysis and then for a further 20 min for analysis with HCS. In a time-matched test, HCS and the manual analysis produced activity data that generated identical conclusions regarding treatment effects and strain differences. Both HCS and the manual analysis found the C57BL/6 controls were overall more active, but not following vasectomy, when both types of analysis detected markedly reduced activity. Low-dose meloxicam (5 mg/kg) had a positive effect on postoperative mobility in the C3H/HeNCrl mice; however, increasing the dose rate progressively reduced this. These effects were also detected with the manual analysis. Overall, HCS provided a sufficiently accurate and rapid method of analysing mouse behaviour encouraging more prolonged assessments in the future. This capability and the possibility of training the software to recognize a greater range of behaviours, including pain-specific indicators, should be of considerable value for assessing postoperative behaviour in both mice and rats. This would allow analgesic requirements to be investigated in a greater range of rodent models than is currently feasible with conventional analysis methods.
Under the 1876 Cruelty to Animals Act it is necessary to recognise pain so that an assessment may be made to determine if it is 'an experiment calculated to give pain' and 'to prevent the animal feeling pain'. Under the conditions of the licence it is also necessary to recognise 'severe pain which is likely to endure' and 'suffering considerable pain'. In the White Paper May 1983 (Command 8883) it is stated that: 'in the application of controls the concept of pain should be applied in a wide sense' and 'the Home Secretary's practice has been to interpret the concept of pain to include disease, other disturbances of normal health, adverse change in physiology, discomfort and distress'. The draft European Convention for the Protection of Vertebrate Animals used for Experimental and other Purposes, aims to control, subject to specific exceptions, any experimental or other scientific procedure which 'may cause pain, suffering, distress or lasting harm'. (The White Paper states that UK control will be stricter than the Council of Europe proposals.) Thus, there is a considerable onus on the experimenter to recognise pain (not to define it) and to alleviate it. It is intended that this article should be of help, not only to newcomers inexperienced in the recognition of pain, but also possibly to those relatively experienced workers who may be called upon to evaluate the pain involved in a new model or an individual animal.(ABSTRACT TRUNCATED AT 250 WORDS)
Laboratory animals are used for regulatory testing to assess the safety, efficacy, and/or potential adverse health effects of new chemicals and products such as vaccines, medicines, food additives, pesticides, and industrial chemicals. Testing results are used for risk assessment decisions intended to safeguard human and animal health. However, chemical toxicity and vaccine testing can cause injury, disease, and mortality involving significant pain and distress. Alleviation of pain and distress in animals during testing is problematic because regulations allow treatment only if the treatment does not interfere with the study. One approach to this problem has been to identify criteria that can serve as the basis for ending a test procedure sooner in an effort to terminate or avoid pain and distress while still allowing attainment of study objectives. These criteria are referred to as humane endpoints because they reduce the severity and/or duration of pain and distress experienced by an animal. New and revised test methods and approaches that incorporate humane endpoints are being considered and adopted by national and international regulatory testing authorities. The prerequisite for adoption of these methods is a determination that the methods have been adequately validated and that they provide equivalent or better information for risk assessment. Further progress in reducing animal pain and distress resulting from regulatory testing is expected as scientific and technological advances are incorporated into testing procedures and strategies.
The use of mice in biomedical research is increasing, largely due to the production and use of genetically engineered animals. Providing postoperative pain control in mice presents many challenges, and long-acting analgesic preparations would be advantageous for this species. A single subcutaneous injection of a liposome-encapsulated (LE) preparation of oxymorphone was compared with multiple injections of buprenorphine or saline in outbred mice undergoing splenectomy. Control groups were given isoflurane alone or isoflurane and an injection of LE oxymorphone but did not undergo surgery. The following parameters were evaluated for 5 days after surgery and were compared with presurgical baseline data for each group: food and water consumption, body weight, ethographic score, and voluntary exercise on a running wheel. Ethographic scores indicated less postsurgical pain in both groups of mice that received either analgesic preparation compared with mice that received only saline. However, mice given LE oxymorphone had superior postoperative recovery, as measured by wheel-running distance and body weight gain, compared with mice given buprenorphine or saline. Mice undergoing splenectomy had significant decreases in body weight, food and water consumption, voluntary exercise, and other normal behaviors. Administration of liposomal oxymorphone at the time of surgery improved postsurgical recovery as measured by these parameters compared with multiple injections of buprenorphine or saline alone. Administration of LE oxymorphone at the time of surgery improved postsurgical recovery, as measured by these parameters.
A shock canine pneumonia model that permitted relief of discomfort with the use of objective criteria was developed and validated. After intrabronchial Staphylococcus aureus challenge, mechanical ventilation, antibiotics, fluids, vasopressors, sedatives, and analgesics were titrated based on algorithms for 96 h. Increasing S. aureus (1 to 8 x 10(9) colony-forming units/kg) produced decreasing survival rates (P = 0.04). From 4 to 96 h, changes in arterial-alveolar oxygen gradients, mean pulmonary artery pressure, IL-1, serum sodium levels, mechanical ventilation, and vasopressor support were ordered based on survival time [acute nonsurvivors (< or =24 h until death, n = 8) > or = subacute nonsurvivors (>24 to 96 h until death, n = 8) > or = survivors (> or =96 h until death, n = 22) (all P < 0.05)]. In the first 12 h, increases in lactate and renal abnormalities were greatest in acute nonsurvivors (all P < 0.05). Compared with survivors, subacute nonsurvivors had greater rises in cytokines and liver enzymes and greater falls in platelets, white cell counts, pH, and urine output from 24 to 96 h (all P < 0.05). Importantly, these changes were not attributable to dosages of sedation, which decreased in nonsurvivors [survivors vs. nonsurvivors: 5.0 +/- 1.0 vs. 3.8 +/- 0.7 ml x h(-1) x (fentanyl/midazolam/ medetomidine)(-1); P = 0.02]. In this model, the pain control regimen did not mask changes in metabolic function and lung injury or the need for more hemodynamic and pulmonary support related to increasing severity of sepsis. The integration into this model of both specific and supportive titrated therapies routinely used in septic patients may provide a more realistic setting to evaluate therapies for sepsis.
Among animals used in research, teaching and testing, mice are now widely recognized as the most important model for human diseases and disorders. They comprise the majority of all experimental mammals and tend to be the model of choice used for research into many diseases/disorders including cancer, heart disease, asthma, Alzheimer's, Down syndrome, deafness, osteoporosis, obesity, diabetes and even mental health research. Additionally the laboratory mouse continues to play a widely publicized vital role in the human genome project. One of the most time-consuming activities in research laboratories is looking up information specific to the species or strain of animal being used. This book, part of the highly successful Handbook of Experimental Animals series, allows the user quick access to any point of interest on the mouse as an experimental model.
IntroductionAnatomic FeaturesBibliography For Introduction and Anatomic FeaturesViral InfectionsBibliography For Viral InfectionsBacterial and Mycotic InfectionsBibliography For Bacterial and Mycotic InfectionsBibliography For Parasitic DiseasesNutritional and Metabolic DisordersBibliography For Nutritional and Metabolic DisordersEnvironmental, Genetic, and Other DisordersBibliography For Environmental, Genetic, and Other DisordersDiseases Associated With AgingBibliography For Diseases Associated With AgingNeoplasmsBibliography For Neoplasms
The quest for humane endpoints to studies involving the use of animals may sound altruistic: The goal is admirable but aside from philosophical implications, the achievement of the goal is essential. Mankind has benefited from research using animals. The safety of the food and drug supplies of the world, as well as other chemicals used in today's societies, has been ensured at significantly higher levels because materials have been evaluated through the use of toxicity testing in animals. There is no doubt that manking has benefited substantially from toxicity testing. Convention on traditions, as applied to the use of animals in testing, are used to generate acceptance of older methods. These methods have been validated and documented as being relatively accurate predictors of hazards to which humans may be exposed. The drawback to the use of traditional methods is that the public, people concerned about animal welfare, and the scientific community realize that change is possible. This concept was not original with Russell and Burch, although the initiation of the 3 Rs and the search for alternatives are most often ascribed to them. The goals of toxicology have not changed; however, they have been suffused with the need for more humane ways to accomplish the same results - proof of safety or hazard from materials to which humans may be exposed. The public is alert to any actions taken by scientists. All appropriate measures must be taken to assure the public that (1) their safety is being considered, and (2) no animals are suffering from needless pain, stress, or distress in efforts to assure this safety. The search for more human means of obtaining applicable data, including refinement of experimental procedures, reduction in the numbers of animals used, or replacement of animals with validated alternatives, must be practived by investigators and enforced by ethical review bodies charged with the approval of animal use protocols.
Published results of a poll taken in the United Kingdom about public views on animal experimentation (Aldhous et al. 1999) reveal that people provided with impartial informa- tion appear to weigh the pros and cons of each experiment carefully before deciding whether they are willing to support the use of animals in research. The results also reveal that potential distress is an important factor in the public's sup- port of specific uses of animals. Death may be used as the experimental endpoint in can- cer research, studies of infectious disease, carcinogenicity and toxicology (e.g., the LD50 test), drug comparisons, and vaccine potency testing (Hamm 1995). In this article, we discuss humane endpoints that can replace lethality as an endpoint in vaccine potency testing.
Food and water restriction protocols are common in animal research, yet they often elicit discussion and controversy among institutional animal care and use committee members who review them. Determining a single standard by which all restriction protocols can be evaluated or performed may not be realistic. However, information about the physiologic and behavioral impact of food and water restriction can provide a basis for making rational judgments about these issues in general. This review will discuss the physiologic and behavioral consequences of food or water deprivation periods of 24 h or less and of chronic restriction schedules, with special reference to protocols that use food or water restriction as a motivational tool for behavioral training.
In 1959, William Russell and Rex Burch published "The Principles of Humane Experimental Technique". They proposed that if animals were to be used in experiments, every effort should be made to Replace them with non-sentient alternatives, to Reduce to a minimum the number of animals used, and to Refine experiments which used animals so that they caused the minimum pain and distress. These guiding principles, the "3 Rs" of animal research, were initially given little attention. Gradually, however, they have become established as essential considerations when animals are used in research. They have influenced new legislation aimed at controlling the use of experimental animals, and in the United Kingdom they have become formally incorporated into the Animal (Scientific) Procedures Act. The three principles, of Replacement, Reduction and Refinement, have also proven to be an area of common ground for research workers who use animals, and those who oppose their use. Scientists, who accept the need to use animals in some experiments, would also agree that it would be preferable not to use animals. If animals were to be used, as few as possible should be used and they should experience a minimum of pain or distress. Many of those who oppose animal experimentation, would also agree that until animal experimentation is stopped, Russell and Burch's 3Rs provide a means to improve animal welfare. It has also been recognised that adoption of the 3Rs can improve the quality of science. Appropriately designed experiments that minimise variation, provide standardised optimum conditions of animals care and minimise unnecessary stress or pain, often yield better more reliable data. Despite the progress made as a result of attention to these principles, several major problems have been identified. When replacing animals with alternative methods, it has often proven difficult to formally validate the alternative. This has proven a particular problem in regulatory toxicology, especially when combined with the labyrinthine processes of the various regulatory authorities. The principle of Reduction would appear less contentious, but its application has highlighted the difficulties of providing appropriate expert statistical advice, especially in academic research facilities. In some instances, concern to implement Reduction strategies can result in the use of too few animals, which leads to inconclusive results, and wasteful experiments. It is in the area of Refinement, however, that major problems have arisen. Much of our judgement of what represents Refinement is based on little more than common sense. We make assumptions about animals and their feelings that often have little scientific basis. In many instances we may be correct, but these assumptions may become incorporated into institutional or national policies, without any attempt to verify them. To give an example - it is reasonable to assume that animals will experience pain after a surgical procedure, so pain-relieving drugs should be given to prevent this. We have some idea of the appropriate dose of analgesics for most animals, but effective pain relief requires that dose given is adjusted to meet the requirements of the individual animal. Requiring every animal to have the same dose of the same drug after any surgical procedure is not the best way of dealing with post-operative pain. Discussion of these problems should not detract from the very significant progress that has been made in the 40 or so years since Russell and Burch set out their guiding principles. What is needed now is greater academic focus on this area, not only to work on new methods of implementing the 3Rs, but also to disseminate current "Best Practice", and to revise this advice as further progress is made.
In this study, we fed a standard NIH-31 diet fortified with vitamin E to C57BL/6 mice and strains of mice with a C57BL/6 background that had spontaneously developed ulcerative dermatitis (UD). In addition to the therapeutic response to increased levels of vitamin E, we also defined the occurrence of UD within our facility in terms of age, sex, coat color, and lesion location on the body. Mice with spontaneous UD were fed a vitamin E-fortified diet (3000 IU/kg) for a period of 8 weeks and entered the study without regard to vendor source, age, sex, coat color, or the site or number of UD lesions. We found that lesions occurred most commonly on the dorsal cervical and scapular regions and spared the ventral abdomen and thorax. No sex or coat color predilection was noted for the development of UD, however males were older than females at the time of lesion development. Of 71 mice, 32 (45%) had complete lesion re-epithelialization with hair regrowth. Complete lesion repair was not influenced by sex, age, or coat color. The average time to complete lesion repair ranged from 2 to 5 weeks, and there was no correlation with sex or coat color. The positive response to vitamin E suggests that protection from oxidative injury may play a role in the resolution of UD lesions and offers veterinarians and investigators a new treatment option with ease of compliance.
To avoid or reduce terminal suffering associated with dying, animal-use protocols associated with high mortality rates often state that animals used in experiments will be euthanatized if they become moribund. However, effective implementation of this strategy depends on the definition of the term moribund. I propose the strategy of defining the moribund state by the use of objective information collected during the implementation of specific experimental paradigms. The value of clinical intervention in dying animals will also be addressed. An objective data-based approach to predicting the imminence of death in the context of specific experimental paradigms could potentially facilitate the implementation of timely euthanasia and thereby could lead to reduced pain, distress, and suffering in research animals.
Laboratory Animal Medicine
- J.G. Fox
- L.C. Anderson
- F.M. Loew
- F.W. Quimby
- Percy