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Additive Effects of Environmental Enrichment and Ketamine on Neuropathic Pain Relief by Reducing Glutamatergic Activation in Spinal Cord Injury in Rats

Authors:
  • Massachusetts General Hospital and Harvard Medical School

Abstract and Figures

Spinal cord injury (SCI) impairs mobility and often results in complications like intractable neuropathic pain. A multi-approach management of this chronic pain condition has been encouraged, but little has been explored of the field. Here, we focus on the effect and underlying mechanism of environmental enrichment (EE), which promotes voluntary social and physical activities, combined with a clinical analgesic, ketamine, on SCI-induced neuropathic pain as well as motor dysfunction. We performed T13 spinal hemisection in rats, which induced unilateral motor impairment and neuropathic pain-like behaviors in the hindlimb. Treatment regimen started a week after SCI, which consists of ketamine administration (30 mg kg–1 day–1; intramuscular) for 10 days, or EE housing for 20 days, or their combination. Paw withdrawal response to mechanical and thermal stimuli, motor function, burrowing behaviors, and body weight was monitored. Spinal segments at T13 lesion and L4–L6 were collected for histopathological and protein analyses. The joint treatment of EE and ketamine provided greater relief of pain-like behaviors and locomotor recovery than did either paradigm alone. These improvements were associated with reduced cavitation area, astrogliosis, and perilesional phosphorylation of glutamate N-methyl-D-aspartate receptor (NMDAR). Concurrently, lumbar spinal analysis of NMDAR-linked excitatory markers in hypersensitization showed reduced activation of NMDAR, mitogen-activated protein kinase (MAPK) family, nuclear factor (NF)-κB, interleukin (IL)-1β signaling, and restored excitatory amino acid transporter 2 level. Our data support a better therapeutic efficacy of the combination, EE, and ketamine, in the attenuation of neuropathic pain and motor recovery by reducing spinal glutamatergic activation, signifying a potential multifaceted neurorehabilitation strategy to improve SCI patient outcome.
| Environmental enrichment (EE) and ketamine (K) reduced nociceptive responses in hindpaws after spinal cord injury (SCI). (A-C) EE (n = 14), K (n = 8), and their combination EEK (n = 9) treatments alleviated SCI-induced mechanical allodynia [paw withdrawal threshold (PWT)]. n = 6 in sham and n = 8 in the SCI group. Two-way repeated-measures ANOVA (effect vs. group × time interaction) followed by Tukey's post hoc test. F ketamine (10,95) = 13.78, F ee (10,125) = 13.65, F eek (10,100) = 16.84. *P < 0.05, **P < 0.01, ***P < 0.001 vs. SCI. (D) Comparison between treatment effects over time by measuring area under the curve (AUC) demonstrated a higher efficacy of the joint treatment than individual ones in combating allodynia. One-way ANOVA (effect vs. group) followed by Tukey's post hoc test. F(3,33) = 78.27. *P < 0.05, **P < 0.01, ***P < 0.001. (E-G) SCI-induced thermal hyperalgesia [paw withdrawal latency (PWL)] was reduced by either ketamine (n = 8) or EE (n = 14) and was reversed by the EEK group (n = 9). n = 6 in sham and n = 7 in the SCI group. Two-way repeated-measures ANOVA (effect vs. group × time interaction) followed by Tukey's post hoc test. F ketamine (10,90) = 5.665, F ee (10,120) = 2.166, F eek (10,95) = 4.218. *P < 0.05, **P < 0.01, ***P < 0.001 vs. SCI. (H) Between-treatment comparison over time (AUC) demonstrated an added benefit of the combined treatment EEK in hyperalgesia relief. AUC is computed from timepoints day 10 to 28, which are after treatment started. One-way ANOVA (effect vs. group) followed by Tukey's post hoc test. F(3,33) = 27.72. *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± standard deviation (SD). Double-end line in bold indicates the 10-day period of ketamine administration (day 8 to 17). Sham, sham-operated group; BL, baseline.
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ORIGINAL RESEARCH
published: 22 March 2021
doi: 10.3389/fnins.2021.635187
Edited by:
Giacinto Bagetta,
University of Calabria, Italy
Reviewed by:
Anna Maria Pittaluga,
University of Genoa, Italy
Jacqueline Sagen,
University of Miami, United States
*Correspondence:
C. W. Cheung
cheucw@hku.hk
Specialty section:
This article was submitted to
Neuropharmacology,
a section of the journal
Frontiers in Neuroscience
Received: 02 December 2020
Accepted: 05 February 2021
Published: 22 March 2021
Citation:
Tai WL, Sun L, Li H, Gu P,
Joosten EA and Cheung CW (2021)
Additive Effects of Environmental
Enrichment and Ketamine on
Neuropathic Pain Relief by Reducing
Glutamatergic Activation in Spinal
Cord Injury in Rats.
Front. Neurosci. 15:635187.
doi: 10.3389/fnins.2021.635187
Additive Effects of Environmental
Enrichment and Ketamine on
Neuropathic Pain Relief by Reducing
Glutamatergic Activation in Spinal
Cord Injury in Rats
W. L. Tai1, L. Sun1,2, H. Li1, P. Gu1, E. A. Joosten1,3,4 and C. W. Cheung1*
1Laboratory and Clinical Research Institute for Pain, Department of Anesthesiology, Queen Mary Hospital, The University
of Hong Kong, Hong Kong, China, 2The First Rehabilitation Hospital of Shanghai, Brain and Spinal Cord Innovation
Research Center, Advanced Institute of Translational Medicine, Tongji University School of Medicine, Shanghai, China,
3Department of Anesthesiology and Pain Management, University Pain Centre Maastricht (UPCM), Maastricht University
Medical Centre, Maastricht, Netherlands, 4Department of Translational Neuroscience, School for Mental Health
and Neuroscience, Maastricht University, Maastricht, Netherlands
Spinal cord injury (SCI) impairs mobility and often results in complications like intractable
neuropathic pain. A multi-approach management of this chronic pain condition has
been encouraged, but little has been explored of the field. Here, we focus on the
effect and underlying mechanism of environmental enrichment (EE), which promotes
voluntary social and physical activities, combined with a clinical analgesic, ketamine,
on SCI-induced neuropathic pain as well as motor dysfunction. We performed
T13 spinal hemisection in rats, which induced unilateral motor impairment and
neuropathic pain-like behaviors in the hindlimb. Treatment regimen started a week after
SCI, which consists of ketamine administration (30 mg kg1day1; intramuscular)
for 10 days, or EE housing for 20 days, or their combination. Paw withdrawal
response to mechanical and thermal stimuli, motor function, burrowing behaviors,
and body weight was monitored. Spinal segments at T13 lesion and L4–L6 were
collected for histopathological and protein analyses. The joint treatment of EE and
ketamine provided greater relief of pain-like behaviors and locomotor recovery than did
either paradigm alone. These improvements were associated with reduced cavitation
area, astrogliosis, and perilesional phosphorylation of glutamate N-methyl-D-aspartate
receptor (NMDAR). Concurrently, lumbar spinal analysis of NMDAR-linked excitatory
markers in hypersensitization showed reduced activation of NMDAR, mitogen-activated
protein kinase (MAPK) family, nuclear factor (NF)-κB, interleukin (IL)-1βsignaling, and
restored excitatory amino acid transporter 2 level. Our data support a better therapeutic
efficacy of the combination, EE, and ketamine, in the attenuation of neuropathic pain
and motor recovery by reducing spinal glutamatergic activation, signifying a potential
multifaceted neurorehabilitation strategy to improve SCI patient outcome.
Keywords: environmental enrichment, ketamine, neuropathic pain, neuroplasticity, neuroprotection, NMDA
receptor, spinal cord injury
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Tai et al. Additive Analgesia by Enriched Environment and Ketamine
INTRODUCTION
Chronic neuropathic pain develops in approximately 65% of
people following spinal cord injury (SCI), severely compromising
patient’s life on top of motor impairment (Siddall et al., 2003;
Duenas et al., 2016). Despite rigorous research effort, available
treatments remain limited and undesirable (Ramer et al., 2014).
Since chronic pain and neurodegeneration are strongly linked
(Costigan et al., 2009), a multitudinal intervention targeting both
complications may result in complementary improvement of
sensory and motor functions in SCI.
Environmental enrichment (EE) is a preclinical model of
rehabilitation that facilitates voluntary motor, sensory, and
cognition activities by provision of a stimulating environment,
avoiding the risk of exercise overload. It is well-documented to
enhance neurogenesis and locomotion, enabling its translational
use in neurorehabilitation unit (Janssen et al., 2012;Tai et al.,
2018b), but its role in post-SCI recovery remains largely
unknown. A few preclinical studies demonstrated that SCI
animals housed in enriched environment have shown reduced
lesion volume and improved sensorimotor recovery (Berrocal
et al., 2007;Koopmans et al., 2012). Moreover, EE has been
proven beneficial in attenuating allodynia and hyperalgesia
in both neuropathic and inflammatory pain models (Gabriel
et al., 2009;Stagg et al., 2011). However, such analgesic effects
remain partial (Berrocal et al., 2007;Koopmans et al., 2012).
In fact, studies of traumatic brain injury have suggested that
combination of EE with selective pharmacotherapies can confer
added benefits (de la Tremblaye et al., 2019).
Ketamine, a classic anesthetic, provides strong analgesia
by blocking the glutamate N-methyl-D-aspartate receptors
(NMDARs) (Bell, 2017). In the recent decade, low dosage of
ketamine has shown distinctive analgesic efficacy in neuropathic
pain, which warrants a favorable safety profile, escalating its
clinical use (Schwartzman et al., 2009;Amr, 2010;Pourmand
et al., 2017). Remarkably, ketamine continues to produce
neuropathic pain-relief when its effective drug level subsided
(Sleigh et al., 2014). It is possible that subanesthetic ketamine
may be an effective adjunct to compensate the limited analgesic
effects of EE.
The combination of EE and ketamine may provide additive
therapeutic benefit by targeting the glutamatergic system,
which is essential in neuroplasticity and pain (Kentner et al.,
2016;Bell, 2017). Dysregulation of glutamate transmission
(i.e., glutamate excitotoxicity and NMDAR overexcitation)
contributes to central sensitization and neurodegeneration in
SCI (Paoletti et al., 2013). EE was shown to reduce spinal
NMDAR phosphorylation following multiple sclerosis (Benson
et al., 2015), and ketamine was found to restrict excitotoxicity
and confer neuroprotection by NMDAR inhibition (Bell, 2017).
Among the NMDAR subtypes, a crucial role of the upregulated
NR2B-containing NMDAR has been highlighted in chronic
pain, but not in acute nor physiological pain (Zhuo, 2009).
NR2B-NMDAR activation has been specifically related to
excitotoxicity and neuronal cell death (Hardingham and Bading,
2010). The current study aimed to investigate the efficacy of
combination of EE and ketamine in attenuating SCI-induced
neuropathic pain and motor defects as well as on NR2B-
NMDAR activity.
MATERIALS AND METHODS
Animal
Adult male Sprague–Dawley rats (250–300 g) were kept
individually in plastic cages with floor covered with soft bedding
at room temperature and maintained on a light/dark cycle of 12-
h day/night. Food and water were provided ad libitum. Animal
experiments were conducted according to the US National
Institute of Health Guide for the Care and Use of Laboratory
Animals and were approved by the Committee on the Use of
Live Animals in Teaching and Research from The University of
Hong Kong (Project #3498-14).
Experimental Design
Animals were randomly assigned to the following: sham, SCI
control (SCI group), SCI plus ketamine (K group), SCI plus EE
(EE group), and SCI plus EE, and ketamine (EEK group). Before
surgery, standard housing was applied to all animals that they
were housed two per cage in a conventional 1291H rat cage
[42.5 cm (L) ×26.6 cm (W) ×18.5 cm (H)] (Lab Animal Unit,
The University of Hong Kong, Hong Kong, China) with nesting
materials (sawdust) only. After surgery, all animals were housed
individually in standard housing condition for a week. Then rats
in the SCI and K groups were kept in the same setting onward,
while rats in the EE and EEK groups were housed three per EE
cage (Figure 1).
Behavioral tests were performed under blinded conditions
preoperatively [baseline (BL)] and on postoperation day (POD)
1, 7, 10, 14, 21, and 28 based on test type (Figure 1C). Of
note, for the K and EEK groups, behavior tests were performed
before ketamine injection when the test day overlapped with
ketamine administration day, to avoid any immediate influence
of ketamine injection.
Environmental Enrichment Housing
The EE housing is adapted from a previous study (Gabriel et al.,
2009) and modified to comply with guidelines of the Lab Animal
Unit of The University of Hong Kong. The EE setup consists of a
paint-coated metallic wire cage [69 cm (L) ×45 cm (W) ×43 cm
(H)] with various objects inside, such as a running wheel [23 cm
(diameter) ×9 cm (platform width) ×26 cm (total height)], a
crawl ball, climbing frames, a tunnel, a jingle ball, and additional
nesting material (Lab Animal Unit) (Figure 1A). Objects in the
cage were renewed once every week. Water and food were placed
at the opposite end of the cage. With this setup, animals had extra
space to explore with moderate, voluntary exercise by walking
back and forth between the water and food and by using the
added attributes.
Spinal Cord Injury Model
T13 hemisection of the spinal cord was performed according
to an established protocol, a moderate SCI model that allows
recovery of basic reflex around POD 7 for pain behavior
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FIGURE 1 | Experimental design. (A) Overview of the standard and enriched environment (EE) housing. (B) Schematic demonstration of T13 spinal cord hemisected
area as covered by dotted red line. Upper and lower sampling sites represent the dorsal and ventral spinal columns respectively where tissue sections for
histopathological analysis were collected. (C) Time course of experimental treatments and behavioral tests. A tube used in the burrowing test is shown in the image.
BL, Baseline; BBB, Basso, Beattie, and Bresnahan locomotor test; PWT, paw withdrawal threshold; PWL, paw withdrawal latency.
assessments (Christensen et al., 1996). In brief, under general
anesthesia with isoflurane (2% for induction and 1% for
maintenance in 70% N2O/30% O2), rats underwent left hemi-
laminectomy at T13 after hair removal and sterilization of the
surgery area with 75% ethanol and Betadine. A small slit was
made in the dura, and the spinal cord was hemisected with
fine iridectomy scissors (FST, Linton, United Kingdom), leaving
intact the dorsal vessel and its major vascular branches. Sham-
operated animal underwent the same surgical process without the
hemisection of the spinal cord.
According to the guidelines provided by the Committee
on the Use of Live Animals in Teaching and Research, the
depth of anesthesia was monitored every 15 min during surgery
by observation of the following: lack of pedal withdrawal to
painful stimulus, heart rate (300–500 beats min1), respiration
rate (70–110 breath min1), saturated pulse oxygenation (i.e.,
mucous membrane color of mouth, pink or pale pink), and body
temperature (37.5–38.5C).
Intramuscular Administration of
Ketamine
Ketamine (Sigma, St. Louis, MO, United States) was dissolved in
saline and injected intramuscularly at a dose of 30 mg kg1day1
for 10 consecutive days starting from POD 8 (Figure 1C). Drug
dosage was adapted from a previous study to obtain a low dose
within the subanesthetic range of ketamine (Goldberg et al., 2005;
Amr, 2010).
Mechanical Allodynia and Thermal
Hyperalgesia
Mechanical and thermal thresholds were evaluated before surgery
and on POD 7, 10, 14, 21, and 28 (Figure 1C) as described
previously (Sun et al., 2018). In brief, animals were placed
individually in plexiglass boxes on a stainless steel mesh floor
(for mechanical test) or on a transparent glass surface (for
thermal test) for at least 15 min to habituate on the test day.
The degree of mechanical allodynia was evaluated by quantifying
paw withdrawal threshold (PWT) of the ipsilateral hindpaw
in response to mechanical stimulation (innocuous) using a
calibrated electronic von Frey filament anesthesiometer (IITC
Life Science, Woodland Hills, CA, United States) with blunted
Von Frey filaments. Animals’ sensitivity to noxious heat was
evaluated using the plantar test, carried out with a paw algesia
meter (IITC). A focused, adjustable, radiant heat light source
beneath a glass floor was applied at the plantar surface of the
ipsilateral hindpaw. Noxious (50% intensity, cutoff time 20 s) heat
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Tai et al. Additive Analgesia by Enriched Environment and Ketamine
stimulus was applied to determine the paw withdrawal latency
(PWL). Three individual readings were taken in an animal with
an interval of at least 5 min and averaged.
Motor Function Assessment
Before injury and on POD 1, 7, 14, 21, and 28, rats were
examined for motor function in an open-field test space using
the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale
(Basso et al., 1995;Figure 1C). In brief, the BBB scale ranges
from 0 (no hindlimb movement) to 21 (normal movement,
including coordinated gait with parallel paw placement). Scores
from 1 to 7 indicate the early phase of recovery with return of
slight to extensive movements in the three joints (hip, knee, and
ankle). Scores from 8 to 13 represent the intermediate phase of
recovery where the return of paw placement and coordinated
movements with the forelimbs is observed. Scores from 14 to
21 show the late phase of recovery with reappearance of toe
clearance during stepping, predominant paw position, trunk
stability, and tail position. Only the scores of the hindlimb
on the hemisected side (ipsilateral side) were shown since
there were no observable differences in locomotion of the
contralateral side.
Burrowing Assay
Rodents are burrowing mammals, and their burrowing ability
is innate, natural, and highly conserved. Therefore, burrowing
assay is considered as an effective measure of overall animal well-
being (Deacon, 2006) and an indirect measure of pain behaviors,
because it does not involve stimulus-evoked pain response (e.g.,
PWT and PWL) (Andrews et al., 2012;Rutten et al., 2014).
The construct of this paradigm for current study is that SCI-
induced neuropathic pain and motor impairment would affect
a rat’s motivation and ability to burrow. Burrowing test was
carried out as previously described (Deacon, 2006) with slight
modifications before surgery and on POD 7 and 28 (Figure 1C).
A burrowing tube (23.5 cm long ×10 cm diameter, Figure 1C)
was raised by two metal stands at the open end to approximately
60 mm higher than the closed end (constructed in-house at The
University of Hong Kong, Hong Kong, China). The tube was
filled with 1-kg pea shingle gravel with a diameter of 2 to 4 mm
(JHC, Hong Kong, China) and placed in a test cage with slightly
sprinkled fresh bedding at the start of each test.
Specifically, all rats received three training sessions as
suggested by previous studies (Andrews et al., 2012;Rutten
et al., 2014) on three consecutive days before the burrowing
performance test. On training day 1, rats were placed in cages
in pairs for 1 h of habituation. Then, an empty tube was placed
into the cage for 1 h to allow familiarization. On days 2 and 3,
the procedure was repeated but with a burrow filled with 1 kg of
gravel. Following the third day of training, rats that demonstrated
a tendency to burrow (90% of those tested) underwent the same
procedure as on the previous day but were tested individually
to determine each rat’s baseline level of burrowing. The amount
of gravel left in the burrow at the end of each test session was
weighted and recorded as a measure of the burrowing behavior.
Animals with burrowing baselines <200 g were excluded from
the experiment (<10%).
Tissue Preparation
At the end of the experiments (POD 28), the spinal cords were
harvested from the experimental animals after euthanization
with sodium pentobarbital. The ipsilateral spinal cord segments
of L4–L6 were removed, snap-frozen in liquid nitrogen,
and stored at 80C until further protein extractions. For
immunofluorescence analysis, spinal segment of T12–T13
centered at the lesion site was dissected from rats perfused with
4% paraformaldehyde (PFA) and then fixed in 4% PFA and
dehydrated in 30% sucrose. Specimens were then cryoprotected
in optimal cutting temperature gel. Twenty-four serial horizontal
cryosections (10 µm thick) were cut from each gray matter-
containing spinal column on the dorsal and ventral sides
(Figure 1B), using a microtome cryostat (Leica Microsystem,
Wetzlar, Germany). Once every four sections in a series of 24
sections was chosen to process for paralleled comparison in
histochemistry studies.
Histopathology and Quantification
Sections were subjected to Nissl staining for assessment of
tissue cavity. The pattern of Nissl-stained neurons allowed the
identification of the spinal dorsal and ventral columns as well
as gray matter preservation. The sections were stained in 0.1%
toluidine blue solution for 3 min and then rinsed briefly in
distilled water followed by differentiation in 95% ethyl alcohol
for 5 min. The boundary of cavitation area was bordered by
the spared tissue. The resulting area was calculated using ImageJ
(National Institutes of Health, Bethesda, MD, United States)
by converting the pixels into millimeters. A total of five tissue
sections centered at the cavitation were analyzed in each animal.
Immunofluorescence and Quantification
Sections were subjected to wash with phosphate-buffered saline
for three times, 5 min each time, before blocking of non-
specific binding. To assess astrocytic reactivity, sections were
stained with astrocyte marker, glial fibrillary acidic protein
(GFAP). For double staining, sections were incubated with
primary antibodies of phosphorylated (p)-NR2B and neuron
marker, NeuN. For triple-labeling, sections were incubated in
p-NR2B antibody with microglia marker, Iba1, and GFAP, or
with Iba1 and NeuN, respectively, at 4C overnight. Details
of primary antibodies are provided in Table 1. Subsequently,
sections were incubated with donkey anti-rabbit, mouse, or goat
secondary antibodies conjugated with Alexa Fluor 488, 568, and
647, respectively (1:1,000, Abcam, Cambridge, United Kingdom).
The slides were cover-slipped in mounting medium containing
DAPI and visualized by Zeiss LSM 780 confocal microscope
(Zeiss, Jena, Germany).
Astrocytic immunoreactivity was semi-quantified by
measurement of immunodensity in an area of 1,800 ×800 µm
centered at the hemisection epicenter using ImageJ (National
Institutes of Health, Bethesda, MD, United States) as previously
described (Sun et al., 2018). The relative immunodensity was
normalized to the corresponding area in the sham group. For
quantification of double-labeled cells, NeuN(+) and NeuN(+)/p-
NR2B(+) cells were counted in the area of 500 µm caudal to
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Tai et al. Additive Analgesia by Enriched Environment and Ketamine
TABLE 1 | Primary antibodies for immunofluorescence.
Antibody Dilution Vendor
Rabbit anti-NMDAR2B,
phospho Tyr 1472
1:100 Merck Millipore, Darmstadt,
Germany
Goat anti-Iba-1 1:50 Novus Biologicals, Littleton, CO,
United States
Mouse anti-NeuN 1:300 Abcam, Cambridge,
United Kingdom
Mouse anti-GFAP 1:250
TABLE 2 | Primary antibodies for western blotting.
Antibody Dilution Vendor
Rabbit anti-phospho-ERK1/2
(p-ERK1/2)
1:1,000 Cell Signaling
Technology, Danvers,
MA, United States
Rabbit anti-ERK1/2 1:4,000
Rabbit anti-phospho-p38 (p-p38) 1:1,000
Rabbit anti-p38 1:4,000
Rabbit anti-phospho-JNK (p-JNK) 1:1,000
Rabbit anti-JNK 1:4,000
Rabbit anti-phospho-NF-κB p65,
Ser536 (p-NF-κB)
1:1,000
Rabbit anti-NF-κB p65 1:4,000
Monoclonal anti-GAPDH 1:4,000
Rabbit anti-phospho-NMDAR2B
Tyr 1472 (p-NR2B)
1:1,000 Merck Millipore,
Darmstadt, Germany
Rabbit anti-NMDAR2B (NR2B) 1:3,000
Rabbit anti-EAAT2 1:3,000 Abcam, Cambridge,
United Kingdom
Rabbit anti-IL-1β1:1,000
the hemisection epicenter. Data were calculated as a percentage
of p-NR2B-positive neurons over the total number of neurons
in the dedicated area. A total of five spinal sections were
analyzed in each animal.
Western Blotting
Frozen spinal cord samples were homogenized (Polytron
Kinematica, Lucerne, Switzerland) in 1 ml of ice-cold lysis
buffer with protease inhibitor cocktails (Sigma, St. Louis,
MO, United States). Clear supernatants were collected after
centrifugation, and protein concentrations were determined by
the Bradford method (Bio-Rad, Hercules, CA, United States).
Protein extracts were subjected to electrophoresis in 10% sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)
gels and subsequently transferred to polyvinylidene fluoride
membranes (Bio-Rad). After being blocked with 5% non-fat milk
for 1 h, membranes were probed with primary antibodies listed
in Table 2 overnight at 4C in incubation buffers. After being
washed with Tris-buffered saline-Tween 20, the membranes
were incubated for 2 h at room temperature in with proper
secondary antibodies horseradish peroxidase (HRP)-linked anti-
rabbit or anti-mouse IgG (Cell Signaling Technology, Danvers,
MA, United States). Protein expressions were detected on films
by enhanced chemiluminescence (Bio-Rad). The relative optical
density of all bands was determined by quantifying the scanned
image with ImageJ software (National Institutes of Health).
Statistical Analysis
All numerical values were stated as mean ±standard deviation
(SD) and analyzed using GraphPad Prism 6 software (GraphPad
Software Inc., La Jolla, CA, United States). Sample size was
estimated based on previous similar studies (de la Tremblaye
et al., 2017;Shi et al., 2018) and our experience (Tai et al.,
2018a). Temporal behavior study and animal body weight were
analyzed using two-way analysis of variance (ANOVA) with
repeated measures followed by Tukey’s post hoc test. Area under
the curve (AUC) and comparison of data from the histological
study and western blot were analyzed with one-way ANOVA
followed by Tukey’s post hoc test. P<0.05 was considered
statistically significant.
RESULTS
Early Onset and Continuous Relief of
Mechanical and Thermal
Hypersensitivities by Joint Treatment of
Environmental Enrichment and Ketamine
in Spinal Cord Injury Rats
Rats developed mechanical allodynia after SCI that persisted
for at least 28 days, which was demonstrated by significantly
lower PWT in the ipsilateral hindpaws of SCI rats than that in
sham (all P<0.001, n= 8, Figures 2A–C). In contrast to the
SCI group, 10 days’ injection of subanesthetic ketamine (30 mg
kg1day1, intramuscular; Figure 2A) significantly increased
PWT by 2 days after initial injection. Although a slight drop
in PWT was seen after ketamine discontinued on POD 17, the
remaining drug effect continued to relieve pain-like behavior till
experiment end point (POD 10 to 28; all P<0.01). EE housing
took effect later than ketamine did but in a progressive manner
(Figure 2B). It first increased PWT by a week after housing
began and continued to further elevate PWT progressively (POD
14 to 28; all P<0.001). In comparison, the combination of
ketamine and EE markedly increased PWT starting from POD
10 (all P<0.001; Figure 2C) and reversed the threshold to basal
level by POD 28 (P= 0.438, EEK: 49.629 ±3.270 g vs. sham:
52.645 ±2.332 g). To evaluate differences in the therapeutic
effects between treatment groups overtime, we compared the
AUC of PWT of treated groups, which is a measure of both
magnitude and duration. Over the experiment time course, the
joint treatment conferred significantly better relief of allodynia
than that by ketamine or EE alone (P<0.01; Figure 2D).
Thermal hyperalgesia manifested as significant decrease of
ipsilateral PWL in response to noxious heat stimulus after SCI
compared with sham (POD 7 to 28, all P<0.01; Figures 2E–
G). After 2 days of ketamine injection (Figure 2E), PWL spiked
as observed on POD 10 (P<0.001, K: 9.987 ±0.702 s vs. SCI:
5.442 ±0.713 s) and returned to the basal level from POD 10
to 21 (all P>0.05 vs. sham). After ketamine cessation (POD
17), such analgesia lasted at least 4 days (POD 21: P<0.001)
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FIGURE 2 | Environmental enrichment (EE) and ketamine (K) reduced nociceptive responses in hindpaws after spinal cord injury (SCI). (A–C) EE (n= 14), K (n= 8),
and their combination EEK (n= 9) treatments alleviated SCI-induced mechanical allodynia [paw withdrawal threshold (PWT)]. n= 6 in sham and n= 8 in the SCI
group. Two-way repeated-measures ANOVA (effect vs. group ×time interaction) followed by Tukey’s post hoc test. Fketamine(10,95) = 13.78, Fee (10,125) = 13.65,
Feek(10,100) = 16.84. *P<0.05, **P<0.01, ***P<0.001 vs. SCI. (D) Comparison between treatment effects over time by measuring area under the curve (AUC)
demonstrated a higher efficacy of the joint treatment than individual ones in combating allodynia. One-way ANOVA (effect vs. group) followed by Tukey’s post hoc
test. F(3,33) = 78.27. *P<0.05, **P<0.01, ***P<0.001. (E–G) SCI-induced thermal hyperalgesia [paw withdrawal latency (PWL)] was reduced by either ketamine
(n= 8) or EE (n= 14) and was reversed by the EEK group (n= 9). n= 6 in sham and n= 7 in the SCI group. Two-way repeated-measures ANOVA (effect vs.
group ×time interaction) followed by Tukey’s post hoc test. Fketamine(10,90) = 5.665, Fee (10,120) = 2.166, Feek(10,95) = 4.218. *P<0.05, **P<0.01, ***P<0.001
vs. SCI. (H) Between-treatment comparison over time (AUC) demonstrated an added benefit of the combined treatment EEK in hyperalgesia relief. AUC is computed
from timepoints day 10 to 28, which are after treatment started. One-way ANOVA (effect vs. group) followed by Tukey’s post hoc test. F(3,33) = 27.72. *P<0.05,
**P<0.01, ***P<0.001. Data are presented as mean ±standard deviation (SD). Double-end line in bold indicates the 10-day period of ketamine administration
(day 8 to 17). Sham, sham-operated group; BL, baseline.
but was unstable and ceased by 11 days post treatment (POD
28: P= 0.660). On the other hand, EE gradually increased
PWL, which reached a significant level by POD 14 (P<0.05,
EE: 7.452 ±1.535 s vs. SCI: 5.432 ±0.724 s). This effect was
maintained throughout the experiment where PWL returned to
the basal level from POD 14 to 21 (all P>0.05 vs. sham,
Figure 2F). In contrast, combined regimen EEK took effect
early and reversed hyperalgesia-like behavior by 6 days after
treatment commenced and returned PWL to the basal level
from POD 10 to 28 (all P>0.05 vs. sham). The observed
significant analgesia lasted through the experiment time course
(POD 14 to 28: all P<0.001 vs. SCI, Figure 2G). Overtime,
the combination treatment demonstrated a strong efficacy against
thermal hyperalgesia, surpassing the effects of ketamine or EE
significantly (P<0.001; Figure 2H).
Environmental Enrichment Combined
With Ketamine Improves Functional
Recovery and Global Well-Being of
Spinal Cord Injury Rats
Rats subjected to SCI surgery lost ipsilateral hindlimb motor
function immediately emerging from anesthesia. Early-to-
intermediate motor recovery phase was observed during the first
week after surgery demonstrated by elevated BBB score from
POD 1 to 7 (Figure 3A). This agreed with previous studies that
used the same spinal cord hemisection model as in this study,
which allows return of intermediate motor reflex for reliable
testing of pain behaviors (Christensen et al., 1996;Coronel et al.,
2011). We observed that only the EEK group achieved full motor
function recovery while others remained at the intermediate-
to-late phase (P<0.05, n= 8, EEK: 20.500 ±0.707 vs. SCI:
14.5 ±2.121). Over the time course, the combined treatment
showed a significantly better effect than that of either K (P<0.05)
or EE (P<0.01) in recovering locomotor after SCI (Figure 3B).
As a global assessment of sensorimotor function and non-
stimulus-evoked pain response, animal’s burrowing behavior
was observed at BL, before and after treatment (POD 7
and 28, respectively). SCI markedly reduced burrowed weight
compared with sham, indicating burrowing behavioral deficit
after injury (POD 7, all P<0.01, Figure 3C). Ketamine
or EE significantly increased the amount of gravel displaced
(POD 28, both P<0.05), while their combination restored
the burrowed amount to the basal level (POD 28, P= 0.995,
EEK: 667.625 ±163.095 g vs. sham: 590.833 ±188.337 g). Of
note, the EEK group burrowed a greater amount than the EE
group (P= 0.030).
In addition to burrowing behavior, animal’s body weight
was also recorded as an evaluation of general well-being. Body
weight of animals in all groups increased gradually over the
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FIGURE 3 | Environmental enrichment (EE) and ketamine (K) promoted motor recovery and general well-being after spinal cord injury (SCI). The combined
treatments of EE and K (EEK) restored the SCI-impaired (A,B) motor function, n= 3 in SCI, n=3inK,n= 4 in EE, and n= 4 in EEK groups [score of Basso, Beattie,
and Bresnahan locomotor test (BBB)]; (C) burrowing ability, n= 6 in sham, n= 6 in SCI, n= 5 in K, n= 8 in EE, and n= 8 in EEK (burrowed gravel weight); and (D–E)
body weight, n= 6 in sham, n= 8 in SCI, n=8inK,n= 14 in EE, and n= 9 in EEK. (A,C,D) Two-way repeated-measures ANOVA (effect vs. group ×time
interaction) followed by Tukey’s post hoc test; (A):F(15,50) = 1.985, (C):F(8,56) = 5.293, (D):F(16,160) = 8.196. *P<0.05, **P<0.01, ***P<0.001. (B,E) Area
under the curve (AUC) is computed from timepoints day 14 to 28, which are after treatment started. One-way ANOVA (effect vs. group) followed by Tukey’s post hoc
test. (B):F(2,9) = 12.65, (E):F(3,33) = 44.99. *P<0.05, **P<0.01, ***P<0.001. Data are presented as mean ±standard deviation (SD). Double-end line in bold
indicates the 10-day period of ketamine administration (day 8 to 17). Sham, sham-operated group; BL, baseline.
experiment course, but the SCI group grew at a significantly
lower rate than the sham group (all P<0.5, Figure 3D). No
significant difference was detected between the EE and SCI
groups. In contrast, from 21 days after SCI, weight growth
in both groups K (1.808 ±0.079%, P= 0.02) and EEK
(1.884 ±0.066%, P<0.01) was significantly greater than that
in the SCI group (1.662 ±0.149%). The weight increase overtime
in the EEK and K groups was greater than that of the EE group
(P<0.001; Figure 3E).
Environmental Enrichment and Ketamine
Reduce Lesion Size and Gliosis in the
Spinal Cord After Injury
To assess the effect of EE and ketamine on SCI lesion size and
astrogliosis, we performed Nissl staining and immunostaining for
GFAP, respectively, on the longitudinal section of the spinal cord
centering at the injury site. Nissl-stained spinal sections identified
the dorsal and ventral tissues by neuronal morphology, which
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FIGURE 4 | Environmental enrichment (EE), ketamine (K), and their combination (EEK) promoted wound heal and reduced gliosis following spinal cord injury (SCI).
Representative sections of the (A) Nissl-stained dorsal and ventral spinal cord were from the same animal in each group to demonstrate an overview of the spinal
cord hemisected site. The dorsal spinal cord can be identified by the dense pattern of small neurons relative to the scattered pattern of large neurons in the ventral.
n= 5/group. (B) Representative images of astrocytic immunoreactivity in the spinal dorsal column. Quantification of Nissl-stained cavitation area in the (C) dorsal and
(D) ventral spinal columns, as well as (E) glial fibrillary acidic protein (GFAP)-stained astrocytic immunoreactivity demonstrated reduced cavitation area and GFAP
reactivity by all treatment groups. n= 3 in sham, n= 3 in SCI, n=4inK,n= 3 in EE, and n= 4 in EEK. One-way ANOVA (effect vs. group) followed by Tukey’s
post hoc test; (C):F(3,16) = 23.82, (D):F(3,16) = 21.77, (E):F(4,12) = 13.66. *P<0.05, **P<0.01, ***P<0.001. Data are presented as mean ±standard
deviation (SD). Cavitation area is bordered by dotted lines.
together revealed a thorough and prominent cavitation on the
hemisected side, resulting in the unilateral sensory and motor
defects (Figure 4A). Tissue surrounding the cavity was a little
deformed but remained intact, indicating a confined injury by the
small incision described in this hemisection model (Christensen
et al., 1996). All treatment groups significantly reduced the spinal
cavitation area in the injured halve compared with the SCI group
(all P<0.001, n=5,Figures 4C,D).
This can also be observed in the lesion area defined by
the GFAP-immunoreactive boundary (Figure 4B). Increased
immunoreactivity of GFAP, a marker of reactive gliosis,
is indicative of neuroinflammation and the formation of
astroglial boundary. Here, all treatment groups reduced GFAP
immunoreactivities along with cavitation size (Figure 4E).
However, the difference between the K and SCI groups (17.8%
reduction, P= 0.546, K: 2.588 ±0.4174% vs. SCI: 3.15 ±0.754%)
was not significant. Both groups EE (68% reduction, P<0.001,
1.005 ±0.2113%) and EEK (52% reduction, P= 0.004,
1.462 ±0.5538%) showed a significantly lower level of astrocytic
reactivity than the SCI group (3.15 ±0.754%) and the K group
(EE vs. K: P= 0.006; EEK vs. K: P= 0.036). Of note, although the
EE group exhibited the lowest GFAP immunoreactivity, its lesion
size appeared larger than that of the other two treatment groups
(38% more than the K group and 63% more than the EEK group).
On the contrary, the EEK group had an intermediate GFAP
reactivity level but the smallest cavitation among the treatment
groups, suggesting that a certain level of astrocytic activity may
benefit wound healing.
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FIGURE 5 | Environmental enrichment (EE) and ketamine (K), especially their combination (EEK), suppressed perilesional activation of neuronal/microglial glutamate
receptor after spinal cord injury (SCI). (A) Representative images and (B) quantification of colocalized phosphorylated N-methyl-D-aspartate receptor subtype 2B
(p-NR2B) with neuronal marker (NeuN). n= 4 in sham, n= 6 in SCI, n=4inK,n= 5 in EE, and n= 5 in EEK. One-way ANOVA (effect vs. group) followed by Tukey’s
post hoc test; B:F(4,19) = 15.72. *P<0.05, **P<0.01, ***P<0.001. Data are presented as mean ±standard deviation (SD). Representative images of
triple-labeled p-NR2B with (C) microglial (Iba-1) and astrocytic [glial fibrillary acidic protein (GFAP)] markers, or with (D) Iba-1 and NeuN. Examples of colocalization
are indicated by white arrow heads.
Environmental Enrichment Combined
With Ketamine Suppresses Perilesional
Activation of Neuronal/Microglial
N-Methyl-D-Aspartate Receptor
To investigate whether the therapeutic effect of EE and ketamine
is associated with neuronal NR2B activation, we double-stained
phosphorylated NR2B (p-NR2B) and the neuronal marker
(NeuN) on the longitudinal section of the dorsal spinal cord
at the injury site. Intense expression of p-NR2B was adjacent
to the lesion after SCI, while moderate-to-low expressions were
seen in the EE, K, and EEK groups (p-NR2B, Figure 5A).
Upon merging with NeuN, colocalization of p-NR2B with
a subpopulation of neuron was detected in the forms of
overlapping or enwrapping parts of the neuronal cell bodies.
This is particularly prominent in the SCI group where p-NR2B-
positive neurons spread around and bordering the lesion.
In comparison, all treatment groups, especially EEK, showed
significantly less p-NR2B-positive neurons (K: P= 0.0052,
EE: P= 0.0372, EEK: P<0.001; n= 5, Figure 5B). Of
note, the EEK group had scattered dot- or short strand-like
expressions of p-NR2B, which resembled those in the sham
group. Interestingly, the morphology of p-NR2B expression may
also indicate involvement of glia in that EEK may reduce glial
activation of NR2B as well.
Hence, we specifically investigated the effect of EEK on
any perilesional activation of glial NMDAR. We triple-labeled
p-NR2B, microglia, and astrocyte in the SCI and EEK groups.
p-NR2B was found to colocalize with microglial cells but not
astrocytes in both the SCI and EEK groups (Figure 5C). However,
highly expressed p-NR2B overlaid with multiple elongated
processes of microglial cells in the SCI group, while a moderate
expression of p-NR2B colocalized with a few oval-like microglial
cells in the EEK group.
Further, triple staining for p-NR2B, microglia, and neuron
revealed active microglial–neuronal interaction in light of
activated NMDAR in both cell types in the SCI group
(Figure 5D). The morphology of p-NR2B expressions highly
resembled active microglia and overlapped with microglial
cell markers exhibiting asymmetrically extended processes that
closely contacted or ensheathed neurons. In contrast, in EEK,
more rounded shapes of microglial cells were observed with
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FIGURE 6 | Environmental enrichment (EE) and (K), especially their combination (EEK), reduced glutamatergic regulation-related markers in spinal cord injury (SCI).
Representative images of protein expressions of phosphorylated (p) (A) NR2B, (B) nuclear factor (NF)-κB, (C) extracellular signal regulated kinase (ERK) 1/2, (D) p38,
(E) c-Jun N-terminal kinase (JNK), and expressions of (F) interleukin (IL)-1βand (G) excitatory amino acid transporter (EAAT) 2. n= 3/group, one-way ANOVA (effect
vs. group) followed by Tukey’s post hoc test; (A):F(4,10) = 80.35, (B):F(4,10) = 9.876, (C):F(4,10) = 18.58, (D):F(4,10) = 20.83, (E):F(4,10) = 21.8,
(F) F(4,10) = 22, (G) F(4,10) = 66.3. *P<0.05, **P<0.01, ***P<0.001. Data are presented as mean ±standard deviation (SD). Sham, sham-operated group.
partial colocalization. No prominent extensions of microglial
processes were recorded.
Combined Environmental Enrichment
and Ketamine Treatment Reduced
Glutamatergic Activation in the Lumbar
Spinal Cord
We examined the glutamate signaling axis in the lumbar spinal
cord, which in part may account for the observed pain-like
behaviors caudal to the injury. After SCI, we detected significantly
increased activation/phosphorylation of NR2B subunit and its
downstream cascade ERK, p38, JNK, and nuclear factor (NF)-
κB (all P<0.01, n= 3, Figures 6A–F). The protein expression
of EAAT2 also decreased significantly (P<0.001, Figure 6G).
These alterations were mitigated by all treatments, but to a greater
degree by the combined treatment, restoring the dysregulated
glutamatergic signaling after SCI.
DISCUSSION
We demonstrated a novel multi-functional regimen, combining
EE housing and ketamine, that effectively improves post-SCI
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Tai et al. Additive Analgesia by Enriched Environment and Ketamine
recovery from both motor and nociceptive defects. The beneficial
effects of the combined treatment were superior to those of
either ketamine or EE alone in locomotor improvement and
neuropathic pain relief. In our SCI model, behavioral deficits
were observed with marked tissue deterioration, astrogliosis,
and activation of neuronal and microglial NR2B subunits
of NMDAR at the spinal lesion site. Further, in line with
the hindpaw allodynia and hyperalgesia, we found augmented
activation of NR2B, MAPK family, NF-κB, and interleukin (IL)-
1β, as well as downregulated EAAT2 expression in the lumbar
spinal cord. These changes were restored by ketamine, EE,
and to a greater extent by their combination as compared
with individual treatments, suggesting an additive effect by the
joint regimen.
Here, evaluation of EE or ketamine as individual treatment has
proven beneficial for post-SCI recovery. Consistent with previous
studies (Berrocal et al., 2007;Koopmans et al., 2012), our EE
paradigm strengthened locomotion and alleviated neuropathic
pain-like behaviors in SCI rats. However, EE treatment alone
has a later onset in both the pain-related behavioral and
motor function assessments. These data suggest that the impact
of EE is slow paced but shows a trend of improvement in
that its effect maybe better observed over a long-term scale.
This agrees with clinical studies of EE in the rehabilitation
unit, which observed better recovery in patients over months
(McDonald et al., 2018). To boost its therapeutic efficacy, we
adopted the subanesthetic use of ketamine and a multiday-
administration scheme, which has been shown to be safe with
distinctive efficacy against chronic pain clinically (Goldberg
et al., 2005;Amr, 2010). In this study, post-drug analgesic effect
of ketamine lasted longer against allodynia (at least 11 days)
than against hyperalgesia (at least 7 days), suggesting that the
post-drug effect was less effective against noxious stimulus-
induced hypersensitivity (hyperalgesia). Although a short-term
ketamine administration would be preferable for patients as
well as to avoid any potential side effects, the analgesic effect
wanes. Therefore, the combination of EE, a long-term feasible
application, and ketamine may optimize each other’s therapeutic
potential. We showed that the joint regimen generated a
profound and prolonged relief of allodynia and hyperalgesia
following SCI even after cessation of ketamine. This recovery
was seen not only in animals’ pain response but also in their
motor function, reflecting a comprehensive advantage of the
combined regimen. In the BBB locomotor assessment, the joint
treatment achieved full score (score of 21) of motor performance
as early as a week after the treatment started. However, the
score in rats treated with ketamine or EE alone plateaued at
the intermediate-to-late stage of recovery (score of 13 to 18),
indicating permanent damage (Basso et al., 1995). Similar to
clinical observation, a spontaneous motor recovery after SCI
can last for years but remains limited (Fakhoury, 2015). Our
finding indicates that the joint treatment of EE and ketamine has
a strong potential in locomotor improvement. The burrowing
test, which evaluates animal’s well-being and non-stimulus-
evoked pain response, also found greater improvement in the
EEK group than the individual treatment, which signified an
additive effect.
From the morphological study, we found that the joint
regimen significantly reduced lesion as shown by quantification
of cavity area and astroglial boarder. Interestingly, the EE
group has the lowest GFAP immunoreactivity among the three
treatment groups but exhibited a slightly larger cavity area than
the other two, suggesting certain wound healing by astroglial
boundary. Although the glial boundary, also termed glial scar
traditionally, was long thought to impede axonal regrowth and
neuromotor recovery, it has an intrinsic protective property in
response to nerve injury. Recent literature strongly supports the
neuroprotective notion of astroglial boarder in that it actually aids
axon regeneration (O’Shea et al., 2017;Bradbury and Burnside,
2019). This might explain the moderate astrocytic reactivity and
optimal lesion closure in the EEK group, suggesting a balanced
act by the joint regimen to optimize tissue recovery. On the other
hand, all three treatments reduced neuronal NR2B (p-NR2B)
activation at the lesion site, but the joint treatment showed a
higher significance in reduction. The sustained overexcitation
of neuron in SCI and chronic pain attributes largely to the
activation of glutamate NMDAR. Selective inhibition of NR2B-
NMDAR produces a significant anti-allodynic effect as well
as neuroprotection (Zhuo, 2009;Woolf, 2011). As EE and
ketamine both suppressed neuronal NR2B-NMDAR activation,
the joint treatment may have combined this effect of the
two and produced the better analgesia and neuroprotection
in this study. This may also explain the overshoot of PWL
in the KEE group after SCI because NMDAR has been
indicated in the maintenance of hyperalgesia, suggesting a strong
anti-hyperalgesic property of this combined treatment (Skyba
et al., 2002). In particular, NR2B-NMDAR is the enriched
subtype at extrasynaptic sites, the interactive points of neuron
with glia, mediating excitotoxicity and pro-death signaling
(Hardingham and Bading, 2010). Potentiated glutamatergic
response in the neuron–microglia interaction was reported
in neuroinflammation-induced hyperalgesia and neuronal cell
death (Kaindl et al., 2012;Sung et al., 2017). To investigate
the perilesional NR2B-NMDAR activity in neuron–microglia
interaction, we triple-labeled p-NR2B with microglia and neuron
markers in the SCI and EEK groups. The morphological
comparison of the two revealed less signs of microglia activation
and microglial–neuronal contacts in the EEK group, as well
as the p-NR2B intensity within them, suggesting that the
noted therapeutic advantages may associate with suppressed
hyperfunction of extrasynaptic NMDAR. On the other hand, we
did not notice colocalization of p-NR2B and astrocyte marker in
the spinal sections. Although astrocytic NMDAR has also been
mentioned in neuropathogenesis, it was reported in the brain but
not the spinal cord (Dzamba et al., 2013).
Besides the injury site, widespread secondary damage
following the initial spinal trauma has been observed in
the lumbar spinal cord and considered as a molecular
basis for below-level neuropathic pain (O’Shea et al., 2017).
Glutamate excitotoxicity plays a crucial role in propagating
the damage by strengthening excitatory neurotransmission via
NMDAR and its downstream MAPK/NF-κB signaling cascade
(Willard and Koochekpour, 2013). In addition, nerve injury
often stimulates release of IL-1βfrom microglia, a signature
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Tai et al. Additive Analgesia by Enriched Environment and Ketamine
of neuroinflammation. It impairs glutamate clearance by
downregulating EAAT2, the major type of glutamate transporter,
but also enhances neuronal release of glutamate, aggravating
glutamate excitotoxicity (Sung et al., 2017). Together, these
events of glutamatergic dysregulation are signature to central
sensitization, which underlies neuropathic pain (Woolf, 2011). In
line with our behavioral observance, the joint regimen restored
these alterations better than did individual treatments. It is
worth noting that a novel effect of EE was shown in inhibiting
lumbar spinal NR2B-NMDAR, consistent with its suppression on
perilesional activation of NR2B, offering an optimal mitigating
potential for SCI.
CONCLUSION
In summary, we demonstrated a novel multitudinal therapeutic
scheme, EE joint with ketamine, which enhanced relief of
SCI-induced neuropathic pain and promoted tissue integrity
as well as locomotion by targeting the glutamatergic system.
Notably, the combined regimen has no adverse effects. But
several limitations in this study should be considered. Although
the spinal hemisection model has been popular in chronic
pain study, this injury rarely occurs in the clinic. Therefore,
findings from this study may be replicated in other SCI
models, such as contusion injury, to merit further translational
investigation. Other potential limitations in the stimulus-evoked
pain assessments (von Frey and Hargreaves) should be noted
that increased hindpaw withdrawal response could result from
hyperreflexia rather than pain after SCI.
While SCI studies that attempt to evaluate multi-approach
therapies and the underlying mechanism remain scarce, our data
may shed light in this field and encourage future investigation of
multi-modal rehabilitations that can minimize secondary damage
and maximize residual function.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included
in the article/supplementary material, further inquiries can be
directed to the corresponding author/s.
ETHICS STATEMENT
The animal study was reviewed and approved by the Committee
on the Use of Live Animals in Teaching and Research, The
University of Hong Kong.
AUTHOR CONTRIBUTIONS
WT and CC designed the study. WT, LS, PG, and HL performed
the experiment. WT, LS, HL, PG, and EJ performed the data
analysis and interpretation. WT wrote the manuscript. WT,
HL, EJ, and CC reviewed and revised the manuscript for final
approval. All authors discussed the results and commented on the
manuscript. All authors contributed to the article and approved
the submitted version.
FUNDING
Support was provided solely from the Department of
Anesthesiology, The University of Hong Kong.
ACKNOWLEDGMENTS
The authors thank Mr. Hoi Chung Shiu (Department of
Anesthesiology, The University of Hong Kong, Hong Kong,
China) for excellent technical support.
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2021 Tai, Sun, Li, Gu, Joosten and Cheung. This is an open-access
article distributed under the terms of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction in other forums is permitted, provided
the original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academicpractice. No
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Frontiers in Neuroscience | www.frontiersin.org 13 March 2021 | Volume 15 | Article 635187
... Glutamate receptor antagonists have been assessed in EAE, primarily in the context of neuroprotection, prevention of demyelination, and improvement of motor deficits (144)(145)(146)(147)(148). Although glutamate receptor antagonists, including memantine (149)(150)(151) or modulators of the glutamatergic system such as ketamine (152,153) have been used for the relief of chronic pain in various diseases or injuries, their effectiveness in the alleviation of neuropathic pain during EAE/MS has not been adequately investigated. ...
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Neuropathic pain inflicts tremendous biopsychosocial suffering for patients worldwide. However, safe and effective treatment of neuropathic pain is a prominent unmet clinical need. Environmental enrichment (EE) is an emerging cost‐effective non‐pharmacological approach to alleviate neuropathic pain and complement rehabilitation care. We present here a review of preclinical studies in ascertaining the efficacy of EE for neuropathic pain. Their proposed mechanisms, including the suppression of ascending nociceptive signaling to the brain, enhancement of descending inhibitory system and neuroprotection of the peripheral and central nervous systems, may collectively reduce pain perception and improve somatic and emotional functioning in neuropathic pain. The current evidence offers critical insights for future preclinical research and the translational application of EE in clinical pain management. This article is protected by copyright. All rights reserved.
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Traumatic brain injury (TBI) is a significant health care issue that affects over ten million people worldwide. Treatment options are limited with numerous failures resulting from single therapies. Fortunately, several preclinical studies have shown that combination treatment strategies may afford greater improvement and perhaps can lead to successful clinical translation, particularly if one of the therapies is neurorehabilitation. The aim of this review is to highlight TBI studies that combined environmental enrichment (EE), a preclinical model of neurorehabilitation, with pharmacotherapies. A series of PubMed search strategies yielded only nine papers that fit the criteria. The consensus is that EE provides robust neurobehavioral, cognitive, and histological improvement after experimental TBI and that the combination of EE with some pharmacotherapies can lead to benefits beyond those revealed by single therapies. However, it is noted that EE can be challenged by drugs such as the acetylcholinesterase inhibitor, donepezil, and the antipsychotic drug, haloperidol, which attenuate its efficacy. These findings may help shape clinical neurorehabilitation strategies to more effectively improve patient outcome. Potential mechanisms for the EE and pharmacotherapy-induced effects are also discussed.
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Aims: We previously demonstrated that intrathecal IL-1β upregulated phosphorylation of p38 mitogen-activated protein kinase (P-p38 MAPK) and inducible nitric oxide synthase (iNOS) in microglia and astrocytes in spinal cord, increased nitric oxide (NO) release into cerebrospinal fluid, and induced thermal hyperalgesia in rats. This study investigated the role of spinal glutamatergic response in intrathecal IL-1β-induced nociception in rats. Methods: The pretreatment effects of MK-801 (5 μg), minocycline (20 μg), and SB203580 (5 μg) on intrathecal IL-1β (100 ng) in rats were measured by behavior, Western blotting, CSF analysis, and immunofluorescence studies. Results: IL-1β increased phosphorylation of NR-1 (p-NR1) subunit of N-methyl-D-aspartate receptors in neurons and microglia, reduced glutamate transporters (GTs; glutamate/aspartate transporter by 60.9%, glutamate transporter-1 by 55.0%, excitatory amino acid carrier-1 by 39.8%; P<.05 for all), and increased glutamate (29%-133% increase from 1.5 to 12 hours; P<.05) and NO (44%-101% increase from 4 to 12 hours; P<.05) levels in cerebrospinal fluid. MK-801 significantly inhibited all the IL-1β-induced responses; however, minocycline and SB203580 blocked the IL-1β-downregulated GTs and elevated glutamate but not the upregulated p-NR1. Conclusion: The enhanced glutamatergic response and neuron-glia interaction potentiate the intrathecal IL-1β-activated P-p38/iNOS/NO signaling and thermal hyperalgesia.
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There is a need for alternative non-opioid analgesics for the treatment of acute, chronic, and refractory pain in the emergency department (ED). Ketamine is a fast acting N-methyl-d-aspartate (NMDA) receptor antagonist that provides safe and effective analgesia. The use of low dose ketamine (LDK) (< 1 mg/kg) provides sub-dissociative levels of analgesia and has been studied as an alternative and/or adjunct to opioid analgesics. We reviewed 11 studies using LDK either alone or in combination with opioid analgesics in the ED. Ketamine was shown to be efficacious at treating a variety of painful conditions. It has a favorable adverse effect profile when given at sub-dissociative doses. Studies have also compared LDK to opioids in the ED. Although ketamine's analgesic effects were not shown to be superior, they were comparable to opioids. LDK has the benefit of causing less respiratory depression. It likely has less wide spread potential for abuse. Nursing protocols for the administration of LDK have been studied. We believe that LDK has the potential to be a safe and effective alternative and/or adjunct to opioid analgesics in the ED. Additional studies are needed to expand upon and determine the optimal use of LDK in the ED.