[Show abstract][Hide abstract] ABSTRACT: Background
The damaging effect of combustion smoke inhalation on the lung is widely reported but information on its effects on the olfactory bulb is lacking. This study sought to determine the effects of smoke inhalation on the olfactory bulb, whose afferent input neurons in the nasal mucosa are directly exposed to external stimuli, such as smoke.Methods
Adult male Sprague-Dawley rats were subjected to combustion smoke inhalation and sacrificed at different time points. Changes in olfactory bulb proteins including vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), Na+-K+-Cl¿ cotransporter 1 (NKCC1), glial fibrillary acidic protein (GFAP), and aquaporin-4 (AQP4) were evaluated by Western blot analysis. In addition, ELISA was conducted for cytokine and chemokine levels, and double immunofluorescence labeling was carried out for GFAP/VEGF, GFAP/AQP4, NeuN/nNOS, GFAP/NKCC1, NeuN/NKCC1, GFAP/Rhodamine isothiocyanate (RITC), and transferase dUTP nick end labeling (TUNEL). Aminoguanidine was administered to determine the effects of iNOS inhibition on the targets probed after smoke inhalation.ResultsThe results showed a significant increase in VEGF, iNOS, eNOS, nNOS, NKCC1, and GFAP expression in the bulb tissues, with corresponding increases in inflammatory cytokines and chemokines after smoke inhalation. Concurrent to this was a drastic increase in AQP4 expression and RITC permeability. Aminoguanidine administration decreased the expression of iNOS and RITC extravasation after smoke inhalation. This was coupled with a significant reduction in incidence of TUNEL¿+¿cells that was not altered with administration of L-NG-nitroarginine methyl ester (L-NAME).Conclusions
These findings suggest that the upregulation of iNOS in response to smoke inhalation plays a major role in the olfactory bulb inflammatory pathophysiology, along with a concomitant increase in pro-inflammatory molecules, vascular permeability, and edema. Overall, these findings indicate that the olfactory bulb is vulnerable to smoke inhalation.
Journal of Neuroinflammation 10/2014; 11(1):176. · 4.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
We reported previously that amoeboid microglial cells in the postnatal rat brain expressed 2¿,3¿-cyclic nucleotide 3¿-phosphodiesterase (CNPase) both in vivo and in vitro; however, the functional role of CNPase in microglia has remained uncertain. This study extended the investigation to determine CNPase expression in activated microglia derived from cell culture and animal models of brain injury with the objective to clarify its putative functions.Methods
Three-day-old Wistar rats were given an intraperitoneal injection of lipopolysaccharide to induce microglial activation, and the rats were killed at different time points. Along with this, primary cultured microglial cells were subjected to lipopolysaccharide treatment, and expression of CNPase was analyzed by real-time reverse transcription PCR and immunofluorescence. Additionally, siRNA transfection was employed to downregulate CNPase in BV-2 cells. Following this, inducible nitric oxide synthase, IL-1ß and TNF-¿ were determined at mRNA and protein levels. Reactive oxygen species and nitric oxide were also assessed by flow cytometry and colorimetric assay, respectively. In parallel to this, CNPase expression in activated microglia was also investigated in adult rats subjected to fluid percussion injury as well as middle cerebral artery occlusion.Results In vivo, CNPase immunofluorescence in activated microglia was markedly enhanced after lipopolysaccharide treatment. A similar feature was observed in the rat brain after fluid percussion injury and middle cerebral artery occlusion. In vitro, CNPase protein and mRNA expression was increased in primary microglia with lipopolysaccharide stimulation. Remarkably, inducible nitric oxide synthase, IL-1ß, TNF-¿, reactive oxygen species and nitric oxide were significantly upregulated in activated BV-2 cells with CNPase knockdown. siRNA knockdown of CNPase increased microglia migration; on the other hand, microglial cells appeared to be arrested at G1 phase.Conclusions
The present results have provided the first morphological and molecular evidence that CNPase expression is increased in activated microglia. CNPase knockdown resulted in increased expression of various inflammatory mediators. It is concluded that CNPase may play an important role as a putative anti-inflammatory gene both in normal and injured brain.
Journal of Neuroinflammation 08/2014; 11(1):148. · 4.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic activation of microglia, the macrophages of the central nervous system (CNS), has been shown to enhance neuronal damage due to excessive release of proinflammatory cytokines and neurotoxic molecules in a number of neurodegenerative diseases. Recent reports showed altered microRNA (miRNA) expression in immune-mediated pathologies, thus suggesting that miRNAs modulate expression of genes involving immune responses. The present study demonstrates that miRNA-200b is expressed in microglia and modulates inflammatory response of microglia by regulating MAPK pathway. miRNA-200b expression was found to be downregulated in activated microglia in vivo (traumatic brain injury rat model) and in vitro. A luciferase assay and loss- and gain-of-function studies revealed c-Jun, the transcription factor of JNK MAPK pathway to be the target of miR-200b. Knockdown of miR-200b in microglia increased JNK activity along with an increase in pro-inflammatory cytokines, iNOS expression and NO production. Conversely, overexpression of miRNA-200b in microglia resulted in a decrease in JNK activity, iNOS expression, NO production and migratory potential of activated microglia. Furthermore, miR-200b inhibition resulted in increased neuronal apoptosis after treatment of neuronal cells with conditioned medium obtained from microglial culture. Taken together, these results indicate that miRNA-200b modulates microglial inflammatory process including cytokine secretion, NO production, migration and neuronal survival.This article is protected by copyright. All rights reserved.
Journal of Neurochemistry 04/2014; · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Burn injuries result in the release of proinflammatory mediators causing both local and systemic inflammation. Multiple organ dysfunctions secondary to systemic inflammation after severe burn contribute to adverse outcome, with the lungs being the first organ to fail. In this study, we evaluate the anti-inflammatory effects of Parecoxib, a parenteral COX-2 inhibitor, in a delayed fluid resuscitation burned rat model. Anaesthetized Sprague Dawley rats were inflicted with 45% total body surface area full-thickness scald burns and subsequently subjected to delayed resuscitation with Hartmann's solution. Parecoxib (0.1, 1.0, and 10 mg/kg) was delivered intramuscularly 20 min after injury followed by 12 h interval and the rats were sacrificed at 6 h, 24 h, and 48 h. Burn rats developed elevated blood cytokines, transaminase, creatinine, and increased lung MPO levels. Animals treated with 1 mg/kg Parecoxib showed significantly reduced plasma level of CINC-1, IL-6, PGEM, and lung MPO. Treatment of 1 mg/kg Parecoxib is shown to mitigate systemic and lung inflammation without significantly affecting other organs. At present, no specific therapeutic agent is available to attenuate the systemic inflammatory response secondary to burn injury. The results suggest that Parecoxib may have the potential to be used both as an analgesic and ameliorate the effects of lung injury following burn.
International journal of inflammation. 01/2014; 2014:972645.
[Show abstract][Hide abstract] ABSTRACT: Microglia, the resident immune cells of the brain, undergo rapid proliferation and produce several proinflammatory molecules and nitric oxide (NO) when activated in neuropathological conditions. Runx1t1 (Runt-related transcription factor 1, translocated to 1) has been implicated in recruiting histone deacetylases (HDACs) for transcriptional repression, thereby regulating cell proliferation. In the present study, Runx1t1 expression was shown to localize in amoeboid microglial cells of the postnatal rat brain, being hardly detectable in ramified microglia of the adult brain. Moreover, a marked expression of Runx1t1was induced and translocated to nuclei in activated microglia in vitro and in vivo. In view of these findings, it was hypothesized that Runx1t1 regulates microglial functions during development and in neuropathological conditions.
siRNA-mediated knockdown of Runx1t1 significantly decreased the expression level of cell cycle-related gene, cyclin-dependent kinase 4 (Cdk4) and proliferation index in activated BV2 microglia. It was also shown that HDAC inhibitor (HDACi) treatment mimics the effects of Runx1t1 knockdown on microglial proliferation, confirming that microglial proliferation is associated with Runx1t1 expression and HDACs activity. Further, Runx1t1 and HDACs were shown to promote neurotoxic effect of microglia by repressing expression of LAT2, L-aminoacid transporter-2 (cationic amino acid transporter, y+ system), which normally inhibits NO production. This was confirmed by chromatin immunoprecipitation (ChIP) assay, which revealed that Runx1t1 binds to the promoter region of LAT2 and this binding increased upon microglial activation. However, the enhanced binding of Runx1t1 to the LAT2 promoter could not repress the LAT2 expression when the BV2 microglia cells were treated with HDACi, indicating that Runx1t1 requires HDACs to transcriptionally repress the expression of LAT2.
In conclusion, it is suggested that Runx1t1 controls proliferation and the neurotoxic effect of microglia by epigenetically regulating Cdk4 and LAT2 via its interaction with HDACs.
PLoS ONE 01/2014; 9(2):e89326. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Studies investigating hyperbaric oxygen treatment (HBOT) to improve outcome in burns have been inconclusive. In this study, we aimed to characterize early thermal burns injury in adult patients with < 40% total body surface area (TBSA) and to determine the effects of HBOT administered within 24 h to 48 h of a burn injury.
Seventeen subjects were randomized into control (n = 9) and HBOT treatment (n = 8) arms. Burn depth, measured by laser Doppler imaging (LDI) and histologically, white blood cell (WBC) count and plasma cytokine inflammatory markers were assessed at 24 h (pre HBOT) and 48 h (post HBOT) post burn, as were immunohistochemistry and microbiology of burns tissue samples at 48 h post burn.
WBC count and serum interleukin (IL)-1β, IL-4, IL-6, IL-10 and interferon-γ were significantly elevated 24 h after burn, but no significant changes in any of these parameters were found with HBOT. HBOT had no significant effect on burn depth. Two HBOT patients and four control patients developed positive bacterial cultures.
Slower than anticipated recruitment resulted in considerably fewer patients than planned being studied. Inflammatory markers were significantly increased at 24 h in patients with < 40% TBSA burn. Early HBOT had no apparent effects on any of the parameters measured in this small pilot study. HBOT may possibly have a broad-spectrum antimicrobial effect worthy of further study. We report our methodology in detail as a possible model for future burns studies.
Journal of the South Pacific Underwater Medicine Society 09/2013; 43(3):157-161. · 0.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effect of primary blast exposure on the brain is widely reported but its effects on the eye remains unclear. Here, we aim to examine the effects of primary blast exposure on the retina.
Adult male Sprague--Dawley rats were exposed to primary blast high and low injury and sacrificed at 24 h, 72 h, and 2 weeks post injury. The retina was subjected to western analysis for vascular endothelial growth factor (VEGF), aquaporin-4 (AQP4), glutamine synthethase (GS), inducible nitric oxide synthase (NOS), endothelial NOS, neuronal NOS and nestin expression; ELISA analysis for cytokines and chemokines; and immunofluorescence for glial fibrillary acidic protein (GFAP)/VEGF, GFAP/AQP4, GFAP/nestin, GS/AQP4, lectin/iNOS, and TUNEL.
The retina showed a blast severity-dependent increase in VEGF, iNOS, eNOS, nNOS, and nestin expression with corresponding increases in inflammatory cytokines and chemokines. There was also increased AQP4 expression and retinal thickness after primary blast exposure that was severity-dependent. Finally, a significant increase in TUNEL+ and Caspase-3+ cells was observed. These changes were observed at 24 h post-injury and sustained up to 2 weeks post injury.
Primary blast resulted in severity-dependent pathological changes in the retina, manifested by the increased expression of a variety of proteins involved in inflammation, edema, and apoptosis. These changes were observed immediately after blast exposure and sustained up to 2 weeks suggesting acute and chronic injury mechanisms. These changes were most obvious in the astrocytes and Muller cells and suggest important roles for these cells in retina pathophysiology after blast.
Journal of Neuroinflammation 07/2013; 10(1):79. · 4.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Following traumatic brain injury (TBI), resultant voids are unable to support injections of suspension treatments, leading to ineffective healing. Moreover, without a structure to support the large defect, the defect site suffers from mechanical instability, which may impair the healing process. Therefore, having a delivery vehicle that can temporarily fill and provide mechanical support to the defect site may alleviate the healing process. In this work, we reported for the first time, the inflammatory response of brain tissue with polycaprolactone (PCL) and PCL-tricalcium phosphate (TCP) scaffolds designed and fabricated for cranial reconstruction. After cranial defects were created in Sprague-Dawley rats, PCL and PCL-TCP scaffolds were implanted for a period of 1 week and 1 month. Following histology and immunofluorescence staining with ionized calcium binding adaptor molecule-1 (IBA-1), glial fibrillary acidic protein (GFAP), nestin and neuronal nuclei (NeuN), results indicated that IBA-1-positive activated microglia were observed across all groups, and declined significantly by 1 month (p<0.05). Interestingly, IBA-1-positive microglia were significantly fewer in the PCL-TCP group (p<0.05), suggesting a relatively milder inflammatory response. A decrease in the number of GFAP-positive cells among all groups over time (>29%) was also observed. Initially, astrocyte hypertrophy was observed proximal to the TBI site (55% in PCL and PCL-TCP groups, 75% in control groups), but it subsided by 1 month. Proximal to the TBI site, nestin immunoreactivity was intense during week 1, and which reduced by 1 month across all groups. NeuN-positive neurons were shrunken proximal to the TBI site (<0.9mm), 32% smaller in PCL-TCP group and 27% smaller in the PCL group. Based on above data indicating the comparatively milder, initial inflammatory response of brain tissue to PCL-TCP scaffolds, it is suggested that PCL-TCP scaffolds have notable clinical advantages as compared to PCL scaffolds.
Tissue Engineering Part A 05/2013; · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction: Traumatic brain injury (TBI) due to blasts by improvised explosive devices (IEDs), is increasingly seen in several countries. It causes various neuropsychological dysfunctions such as attention deficit, working function, motor skills etc in both animals and humans. Susceptibility weighted imaging (SWI) [1, 2] allows improved detection of paramagnetic hemorrhagic blood components based on their magnetic susceptibility effects. SWI is sensitive to venous blood and can be used to visualise the micro vessels, which is useful in the analysis of TBI. SWI with multiple echoes yields good signal to noise ratio (SNR) as well as contrast-to-noise ratio (CNR)  . In the present study, we explore the blast induced TBI using multi-echo SWI on rodent model. Methods: Blast Test Set-up 5 kg of 2,4,6-trinitrotoluene (TNT) with a penta-erythritol tetra-nitrate (PETN) booster was detonated at 1 m height in each blast. Blast sensors were used to monitor the intensity and duration of blast over pressure (BOP) exposure. Metal cages along with the pressure transducer were set up at 2 m and 3 m alternately from the blast source. All the animals were randomly grouped into 1) Sham: where the subjects were not exposed to blast but anaesthetized; 2) High Blast (HB): where subjects were exposed to a single blast at ~480 kPa BOP at 2 m from blast source; 3) Low Blast (LB): where subjects were exposed to a single blast at ~180 kPa at 3 m from the blast source  . MRI Data Acquisition SWI and high resolution MPRAGE images were acquired on 7T ClinScan (Bruker BioSpin, Germany) equipped with 4 channel RAPID phased array coil before blast (Baseline, BL), on day 1, 3, 5, 14 and 28 after blast (# HB Rat = 6, #LB Rat = 6). Multi echo SWI was performed using TR/flip angle/slice thickness/#slice/FOV/matrix size/# average = 45 ms /20º/0.8 mm/64/36 mm × 27 mm/512×384/2. A total of 5 echoes with a central-echo time of 14.95 ms and an echo-spacing of 4.008 ms (the 5 acquired echo times used were: 6.79, 10.87, 14.95, 19.03, 23.11 ms) were acquired. MPRAGE images were acquired with TR/TE/flip angle/slice thickness/#slice/FOV/matrix size/#average = 2000 ms/1.6 ms/20º/0.5 mm/52/35 mm × 28.44 mm/256×208 (zero filled to 512 × 512)/2.Western blot for Hypoxia-inducible factor (HIF)-1a was also performed to see the changes in deoxygenation at cortex. A Java based ImageJ (National Institute of Health, USA) plugin was developed to quantify the R2* maps of the entire brain. The 5 echo data were fit to mono-exponential to obtain the R2* maps  . Results: Minimum intensity projection (mIP) over 7 slices of 1 st and 2 nd echo at different time points are shown in Fig. 1 (top and middle rows respectively). The filtered phase images of 2 nd echo at different time points are shown in the Fig. 1 (bottom). Calculated R2* map at day 3 is shown in the Fig. 1 (last image of bottom row). The phase profiles of vessels along the red line on phase images in Fig. 1 are shown in the Fig. 2. The largest jump is shown at day 1 and day 3 and the smallest is in BL. The decrease in signal in veins after blast is indicated by arrows (red and purple) in Fig 1. The arrows show the injury and dilation of vessels during the acute phase and recovery in the later phase. The smaller venous structures are better visualized in the 2 nd echo (Fig.1 middle row).Western blot graph of HIF-1a for blast cortex is shown in Fig 3 which shows the significant change in de-oxygenation after blast. Conclusions and Discussions: MPRAGE images at different time points showed no visible trauma where as the SWI show the change in veins in Fig. 1  . Multi-echo SWI provides phase and R2* with better SNR and CNR, making it a sensitive technique to image blast induced TBI [3, 7] . SWI at longer TE has stronger susceptibility effect yielding greater venous contrast, whereas SWI at shorter TE provides higher SNR and less off-resonance artefact  . Larger veins are clearly shown at shorter TE, and smaller venous structures are better visualized at longer TE as in shown in Fig. 1  . Multi-echo SWI may help to detect and classify the types and patters of blast induced TBI. The decrease in the signal in the veins after blast is due to the increase in deoxyhomoglobin  . The HIF-1a result shows the same fact. From the phase profiles in Fig. 2, we can conclude that relative change in cerebral blood flow (CBF) in the post blast trauma is higher than compared to sham [6, 8] . R2* maps calculated from multi-echo SWI data may be used to measure of iron deposition in the brain. Also the calcification and haemorrhage can be easily differentiated with the help of filtered phase images. Overall, the multi-echo SWI is sensitive and very useful technique for TBI imaging and analysis [6, 7, 8] . Fig. 3. Western blot graph of HIF-1a for blast cortex. Fig. 1. mIP of 1 st (top) and 2 nd (middle) echo over 7 slices at different time points for LB. The darkening in the veins post blast is indicted by the arrows. The filter phase images (bottom) at different time points. R2* map (last image at bottom) at day 3.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Hypoxia induces microglial activation which causes damage to the developing brain. Microglia derived inflammatory mediators may contribute to this process. Toll-like receptor 4 (TLR4) has been reported to induce microglial activation and cytokines production in brain injuries; however, its role in hypoxic injury remains uncertain. We investigate here TLR4 expression and its roles in neuroinflammation in neonatal rats following hypoxic injury. METHODS: One day old Wistar rats were subjected to hypoxia for 2 h. Primary cultured microglia and BV-2 cells were subjected to hypoxia for different durations. TLR4 expression in microglia was determined by RT-PCR, western blot and immunofluorescence staining. Small interfering RNA (siRNA) transfection and antibody neutralization were employed to downregulate TLR4 in BV-2 and primary culture. mRNA and protein expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta) and inducible nitric oxide synthase (iNOS) was assessed. Reactive oxygen species (ROS), nitric oxide (NO) and NF-kappaB levels were determined by flow cytometry, colorimetric and ELISA assays respectively. Hypoxia-inducible factor-1 alpha (HIF-1alpha) mRNA and protein expression was quantified and where necessary, the protein expression was depleted by antibody neutralization. In vivo inhibition of TLR4 with CLI-095 injection was carried out followed by investigation of inflammatory mediators expression via double immunofluorescence staining. RESULTS: TLR4 immunofluorescence and protein expression in the corpus callosum and cerebellum in neonatal microglia were markedly enhanced post-hypoxia. In vitro, TLR4 protein expression was significantly increased in both primary microglia and BV-2 cells post-hypoxia. TLR4 neutralization in primary cultured microglia attenuated the hypoxia-induced expression of TNF-alpha, IL-1beta and iNOS. siRNA knockdown of TLR4 reduced hypoxia-induced upregulation of TNF-alpha, IL-1beta, iNOS, ROS and NO in BV-2 cells. TLR4 downregulation-mediated inhibition of inflammatory cytokines in primary microglia and BV-2 cells was accompanied by the suppression of NF-kappaB activation. Furthermore, HIF-1alpha antibody neutralization attenuated the increase of TLR4 expression in hypoxic BV-2 cells. TLR4 inhibition in vivo attenuated the immunoexpression of TNF-alpha, IL-1beta and iNOS on microglia post-hypoxia. CONCLUSION: Activated microglia TLR4 expression mediated neuroinflammation via a NF-kappaB signaling pathway in response to hypoxia. Hence, microglia TLR4 presents as a potential therapeutic target for neonatal hypoxia brain injuries.
Journal of Neuroinflammation 02/2013; 10(1):23. · 4.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neuroinflammation mediated by the activated microglia is suggested to play a pivotal role in the pathogenesis of hypoxic brain injury; however, the underlying mechanism of microglia activation remains unclear. Here, we show that the canonical Notch signaling orchestrates microglia activation after hypoxic exposure which is closely associated with multiple pathological situations of the brain. Notch-1 and Delta-1 expression in primary microglia and BV-2 microglial cells was significantly elevated after hypoxia. Hypoxia-induced activation of Notch signaling was further confirmed by the concomitant increase in the expression and translocation of intracellular Notch receptor domain (NICD), together with RBP-Jκ and target gene Hes-1 expression. Chemical inhibition of Notch signaling with N-[N-(3,5-difluorophenacetyl)-1-alany1- S-phenyglycine t-butyl ester (DAPT), a γ-secretase inhibitor, effectively reduced hypoxia-induced upregulated expression of most inflammatory mediators. Notch inhibition also reduced NF-κB/p65 expression and translocation. Remarkably, Notch inhibition suppressed expression of TLR4/MyD88/TRAF6 pathways. In vivo, Notch signaling expression and activation in microglia were observed in the cerebrum of postnatal rats after hypoxic injury. Most interestingly, hypoxia-induced upregulation of NF-κB immunoexpression in microglia was prevented when the rats were given DAPT pretreatment underscoring the interrelationship between Notch signaling and NF-κB pathways. Taken together, we conclude that Notch signaling is involved in regulating microglia activation after hypoxia partly through the cross talk between TLR4/MyD88/TRAF6/NF-κB pathways. Therefore, Notch signaling may serve as a prospective target for inhibition of microglia activation known to be implicated in brain damage in the developing brain.
PLoS ONE 01/2013; 8(11):e78439. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AIMS: The effect of combustion smoke inhalation on the respiratory system is widely reported but its effects on the central nervous system remains unclear. Here, we aimed to determine the effects of smoke inhalation on the cerebellum and hippocampus which are areas vulnerable to hypoxia injury. METHODS: Adult male Sprague Dawley rats were subjected to combustion smoke inhalation and sacrificed at 0.5, 3, 24 and 72 h after exposure. The cerebellum and hippocampus were subjected to western analysis for VEGF, iNOS, eNOS, nNOS and AQP-4 expression; ELISA analysis for cytokine and chemokine levels; and immunohistochemistry for GFAP/AQP-4, RECA-1/RITC and TUNEL. Aminoguanidine (AG) was administered to determine the effects of iNOS after smoke inhalation. RESULTS: Both the cerebellum and hippocampus showed a significant increase in VEGF, iNOS, eNOS, nNOS and AQP-4 expression with corresponding increases in inflammatory cytokines and chemokines and increased AQP-4 expression and RITC permeability after smoke exposure. AG was able to decrease the expression of iNOS, followed by VEGF, eNOS, nNOS, RITC and AQP-4 after smoke exposure. There was also a significant increase in TUNEL+ cells in the cerebellum and hippocampus which was not significantly reduced by AG. Beam walk test revealed immediate deficits after smoke inhalation which was attenuated with AG. CONCLUSION: The findings suggest that iNOS plays a major role in the CNS inflammatory pathophysiology after smoke inhalation exposure with concomitant increase in pro-inflammatory molecules, vascular permeability and oedema for which the cerebellum appears to be more vulnerable to smoke exposure than the hippocampus.
Neuropathology and Applied Neurobiology 10/2012; · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Diva (death inducer binding to vBcl-2 and Apaf-1)/BclB is a Bcl-2 family member, which is known for its function in apoptosis. Diva/BclB has been shown to interact with NDPKB/Nm23H2, which is involved in cellular differentiation. Thus far, there has been no direct evidence of Diva/BclB having a role in differentiation. In the present study, we investigated the expression of Diva/BclB and NDPKB/Nm23H2 during differentiation in PC- 12 cell line. RESULTS: Our results show that after differentiation, Diva/BclB expression was decreased and reciprocally, NDPKB/Nm23H2 expression was increased and it translocated into the nucleus. Overexpression of NDPKB/Nm23H2 promoted PC-12 neuronal differentiation by increasing neurite outgrowth and arresting cell cycle progression. There was a concurrent downregulation of Diva/Boo when NDPKB/Nm23H2 was overexpressed, which mirrors the effect of NGF on PC-12 cell differentiation. Overexpression of Diva/BclB did not change the expression level of NDPKB/Nm23H2, but inhibited its nuclear localization. Cells that overexpressed Diva/BclB presented a decreased percentage of differentiated cells and average neurite length was shortened. This was due to an increase in the formation of Diva/BclB and NDPKB/Nm23H2 complexes as well as Diva/BclB and beta-tubulin complexes. Concomitantly, there was a decrease in formation of NDPKB/Nm23H2 and beta-tubulin complexes. Overexpression of Diva/BclB also resulted in a higher percentage of S-phase cells. CONCLUSION: Our results showed a novel role for Diva/BclB in neuronal differentiation. Its downregulation during neuronal differentiation may be necessary to allow NDPKB/Nm23H2 and beta-tubulin interaction that promotes NDPKB/Nm23H2 mediated differentiation.
[Show abstract][Hide abstract] ABSTRACT: Microglia, the resident immune cells of the central nervous system (CNS), have two distinct phenotypes in the developing brain: amoeboid form, known to be amoeboid microglial cells (AMC) and ramified form, known to be ramified microglial cells (RMC). The AMC are characterized by being proliferative, phagocytic and migratory whereas the RMC are quiescent and exhibit a slow turnover rate. The AMC transform into RMC with advancing age, and this transformation is indicative of the gradual shift in the microglial functions. Both AMC and RMC respond to CNS inflammation, and they become hypertrophic when activated by trauma, infection or neurodegenerative stimuli. The molecular mechanisms and functional significance of morphological transformation of microglia during normal development and in disease conditions is not clear. It is hypothesized that AMC and RMC are functionally regulated by a specific set of genes encoding various signaling molecules and transcription factors.
To address this, we carried out cDNA microarray analysis using lectin-labeled AMC and RMC isolated from frozen tissue sections of the corpus callosum of 5-day and 4-week old rat brain respectively, by laser capture microdissection. The global gene expression profiles of both microglial phenotypes were compared and the differentially expressed genes in AMC and RMC were clustered based on their functional annotations. This genome wide comparative analysis identified genes that are specific to AMC and RMC.
The novel and specific molecules identified from the trancriptome explains the quiescent state functioning of microglia in its two distinct morphological states.
[Show abstract][Hide abstract] ABSTRACT: While OX42(+) microglia/macrophages have been considered as a scavenger in the brain, NG2(+) cells are generally considered as oligodendrocyte progenitor cells or function-unknown glial cells. Recent evidence showed that under some pathological conditions, certain cells have become positive for both anti-NG2 and anti-OX42 antibodies. Our results suggested that some OX42(+) microglia or macrophages were induced to express NG2 proteins 3 and 5 days later after focal injection of lipopolysaccharide into the brain cortex of Sprague-Dawley rats. In consideration of the induction of NG2 expression may associate with gaining or losing functions of microglia/macrophages, we further showed that, while OX42(+) or ED1(+) microglia/macrophages presented active phagocytic function, NG2(+) /OX42(+) cells failed to engulf latex beads. The induced expression of NG2 protein may possibly indicate the functional diversity of activated microglia/macrophages in the brain.
[Show abstract][Hide abstract] ABSTRACT: Traumatic brain injury (TBI) is a major public-health problem for which mild TBI (MTBI) makes up majority of the cases. MTBI is a poorly-understood health problem and can persist for years manifesting into neurological and non-neurological problems that can affect functional outcome. Presently, diagnosis of MTBI is based on symptoms reporting with poor understanding of ongoing pathophysiology, hence precluding prognosis and intervention. Other than rehabilitation, there is still no pharmacological treatment for the treatment of secondary injury and prevention of the development of cognitive and behavioural problems. The lack of external injuries and absence of detectable brain abnormalities lend support to MTBI developing at the cellular and biochemical level. However, the paucity of suitable and validated non-invasive methods for accurate diagnosis of MTBI poses as a substantial challenge. Hence, it is crucial that a clinically useful evaluation and management procedure be instituted for MTBI that encompasses both molecular pathophysiology and functional outcome. The acute microenvironment changes post-MTBI presents an attractive target for modulation of MTBI symptoms and the development of cognitive changes later in life.
Brain research bulletin 03/2012; 87(4-5):359-72. · 2.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mild traumatic brain injury (mTBI) is one of the most common neuronal insults and can lead to long-term disabilities. mTBI occurs when the head is exposed to a rapid acceleration-deceleration movement triggering axonal injuries. Our limited understanding of the underlying pathological changes makes it difficult to predict the outcome of mTBI. In this study we used a scalable rat model for rotational acceleration TBI, previously characterized for the threshold of axonal pathology. We have analyzed whether a TBI just above the defined threshold would induce any detectable behavioral changes and/or changes in serum biomarkers. The effect of injury on sensory motor functions, memory and anxiety were assessed by beam walking, radial arms maze and elevated plus maze at 3-7 days following TBI. The only behavioral deficits found were transient impairments in working and reference memory. Blood serum was analyzed at 1, 3, and 14 days after injury for changes in selected protein biomarkers. Serum levels of neurofilament heavy chain and Tau, as well as S100B and myelin basic protein showed significant increases in the injured animals at all time points. No signs of macroscopic injuries such as intracerebral hematomas or contusions were found. Amyloid precursor protein immunostaining indicated axonal injuries at all time points analyzed. In summary, this model mimics some of the key symptoms of mTBI, such as transient memory impairment, which is paralleled by an increase in serum biomarkers. Our findings suggest that serum biomarkers may be used to detect mTBI. The model provides a suitable foundation for further investigation of the underlying pathology of mTBI.
[Show abstract][Hide abstract] ABSTRACT: Diva is a member of the Bcl2 family but its function in apoptosis remains largely unclear because of its specific expression found within limited adult tissues. Previous overexpression studies done on various cell lines yielded conflicting conclusions pertaining to its apoptotic function. Here, we discovered the expression of endogenous Diva in PC12 neuronal-like cell line and rat bone marrow mesenchymal stem cells (BMSCs), leading to their utilisation for the functional study of Diva. Through usage of recombinant Fas ligand, hydrogen peroxide, overexpression and knock down experiments, we discovered that Diva plays a crucial pro-survival role via the mitochondrial death pathway. In addition, immunoprecipitation studies also noted a decrease in Diva's interaction with Bcl2 and Bax following apoptosis induced by oxidative stress. By overexpressing Diva in BMSCs, we had observed an increase in the cells' capacity to survive under oxidative stress and microglial toxicity. The result obtained from our study gives us reason to believe that Diva plays an important role in controlling the survival of BMSCs. Through overexpression of Diva, the viability of these BMSCs may be boosted under adverse conditions.
PLoS ONE 01/2012; 7(8):e43180. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A penetrating traumatic brain injury (pTBI) occurs when an object impacts the head with sufficient force to penetrate the skin, skull, and meninges, and inflict injury directly to the brain parenchyma. This type of injury has been notoriously difficult to model in small laboratory animals such as rats or mice. To this end, we have established a novel non-fatal model for pTBI based on a modified air rifle that accelerates a pellet, which in turn impacts a small probe that then causes the injury to the experimental animal's brain. In the present study, we have focused on the acute phase and characterized the tissue destruction, including increasing cavity formation, white matter degeneration, hemorrhage, edema, and gliosis. We also used a battery of behavioral models to examine the neurological outcome, with the most noteworthy finding being impairment of reference memory function. In conclusion, we have described a number of events taking place after pTBI in our model. We expect this model will prove useful in our efforts to unravel the biological events underlying injury and regeneration after pTBI and possibly serve as a useful animal model in the development of novel therapeutic and diagnostic approaches.
Journal of neurotrauma 12/2011; 29(6):1219-32. · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Blast injury to the brain is one of the major causes of death and can also significantly affect cognition and physical and psychological skills in survivors of blast. The complex mechanisms via which blast injury causes impairment of cognition and other symptoms are poorly understood. In this study, we investigated the effects of varying degrees of primary blast overpressure (BOP; 80 and 200 kPa) on the pathophysiological and magnetic resonance imaging (MRI) changes and neurocognitive performance as assessed by the monkey Cambridge Neuropsychological Test Automated Battery (mCANTAB) in non-human primates (NHP). The study aimed to examine the effects of neurobehavioral and histopathological changes in NHP. MRI and histopathology revealed ultrastructural changes in the brain, notably in the Purkinje neurons in the cerebellum and pyramidal neurons in the hippocampus, which were most vulnerable to the blast. The results correlated well with the behavioral changes and changes in motor coordination and working memory of the affected monkeys. In addition, there was white matter damage affecting myelinated axons, astrocytic hypertrophy, and increased aquaporin-4 (AQP-4) expression in astrocytes, suggesting cerebral edema. Increased apoptosis appeared to involve astrocytes and oligodendrocytes in the animals following blast exposure. The small sample size could have contributed to the non-significant outcome in cognitive performance post-blast and limited quantitative analyses. Nevertheless, the study has provided initial descriptive changes for establishing a primary BOP threshold for brain injury to serve as a useful platform for future investigations that aim to estimate brain injury potential and set safe limits of exposure.
Journal of neurotrauma 06/2011; 29(7):1434-54. · 4.25 Impact Factor