Activation of GABA A receptors enhances the behavioral recovery but not axonal sprouting in ischemic rats

Abstract

Background:

GABA_A receptors modulate the behavioral recovery encountered in both experimental animals and patients with ischemic injury, possibly through promoting structural plasticity. We hypothesized that activation of GABA_A receptors would regulate axonal growth, which in turn would improve the behavioral recovery in ischemic rats.

Objective:

To investigate the effects of muscimol on axonal growth, synaptic plasticity and behavioral performance in rats after a focal ischemia induced by endothelin-1 (ET-1).

Methods:

Focal ischemic infarct was induced by ET-1. The rats were randomly divided into 3 groups: sham-operated group, ischemic group, ischemic+muscimol group. The muscimol infusion into contralateral cortex started on post-operative day 7 continuing until day 21. Biotinylated dextran amine was injected on post-operative day 14 into the contralesional motor cortex to trace the crossing corticospinal tract fibers. The expression levels of growth inhibitors, Nogo receptor, NogoA, RhoA, and Rho-associated kinase were measured in the peri-infarct cortex. The expressions of vGlut-1 and postsynaptic density-95 were measured by immunohistochemistry and Western blot in the denervated spinal cord. The behavioral recovery was evaluated by sensorimotor tests on post-operative days 32–34.

Results:

Treatment with the specific GABA_A receptors agonist, muscimol, did not increase axonal growth into the denervated hemispheres and spinal cord after stroke. However, the activation of GABA_A receptors partially improved the rats’ behavioral performance after the ET-1-induced stroke.

Conclusions:

Our study revealed that infusion of muscimol into the contralateral motor cortex during the repair stage could partially improve the behavioral performances without promoting axonal growth from uninjured hemisphere motor cortex to the denervated striatum and spinal cord, nor did it prevent the expression of axonal growth inhibitors in peri-lesioned cortex. More detailed studies will be required to clarify the role of GABA_A Rs in regulating the behavioral recovery after a stroke.

Keywords

Muscimol axonal growth stroke behavioral recovery

1. Introduction

Stroke has been the second major cause of disability and mortality around the world (Varghese et al., 2016). Although thrombolytic therapy is effective and has been added to the standard of care for stroke patients, it has a narrow time window i.e. within 4–6 hours and only 10% of patients are eligible for thrombolysis (Derex et al., 2002; Segura et al., 2008). Unfortunately, there are limited medical treatment options available for the patients beyond the acute stage.

It has been observed that stroke is followed by enhanced spontaneous repair which continues for many weeks; this involves neurogenesis, axonal growth, and oligodendrocyte regeneration (Cramer, 2018; Overman & Carmichael, 2014). Thus, promoting the recovery of neural systems could represent an alternative treatment approach for stroke patients, especially as it would have a wider treatment time window(Cramer, 2018).

γ-Aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the mammalian central nervous system (CNS) (De Luka et al., 1998). The inhibitory effect of GABA on neural cell membrane is primarily mediated by GABA type A receptors (GABA_A Rs) (Kaila et al., 2014). GABA_A Rs are located both in the synapse and extrasynaptically (Glykys & Mody, 2006; Walker & Semyanov, 2008). Muscimol, an agonist at GABA_A Rs, acts similarly at all of the different subunit combinations of GABA_A Rs with the exception of α4β3δ receptors, which are localized in the extrasynaptic space (Ebert et al., 1997; Mi et al., 2008). Therefore, muscimol appears to be a potent agonist targeting both synaptic GABA_A Rs (mediating phasic inhibition) and extrasynaptic GABA_A Rs (mediating tonic inhibition).

The spontaneous repair-related changes occurring in brain after a stroke are not only limited to the perilesional tissue, but also are encountered within the cortralesional hemisphere (Dijkhuizen et al., 2012; Jones et al., 1996; Kawamata et al., 1997). Evidence for the reducing inhibition in the contralateral motor cortex have been obtained in different rat ischemic models (Buchkremer-Ratzmann et al., 1996; Que et al., 1999). In addition, the expressions of different subunits of GABA_A Rs were noted to be decreased in the contralateral motor cortex after ischemic stroke (Lee et al., 2011; Redecker et al, 2000).

It has been claimed that zolpidem, a GABA_A Rs positive allosteric modulator with its highest affinity for the α1 subunit, can promote recovery during the acute stage of experimental stroke i.e. in both the distal middle cerebral artery occlusion model and the photothrombotic model (Hiu et al., 2016). However, little is known about the effect of GABA inhibition obtained by muscimol infusion into the contralateral motor cortex on post-stroke repair (Lee et al., 2011; Mansoori et al., 2014). Mansoori et.al reported that muscimol could promote the behavioral performance of stroke rats. In contrast, after a traumatic cortical injury it has been claimed that an infusion of muscimol into the contralateral motor cortex inhibited the long-term behavioral recovery in mice (Lee et al., 2011). There are only a few studies examining the morphologic changes induced by elevated GABA inhibition in the contralateral motor cortex of ischemic rats (Lee et al.,2011).

Cortical spinal tract (CST) axons from the contralateral motor cortex sprout and cross over the midline into the denervated side following a unilateral stroke (Benowitz & Carmichael, 2010; Zai et al., 2009; Zhao et al, 2013). However, it is well documented that several intrinsic myelin–associated neurite growth inhibitors, including Nogo-A, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, limit axonal growth and plasticity (Benowitz & Carmichael, 2010; Lee et al., 2004). NogoA is one of the major inhibitory factors in CNS and could reduce the extent of spontaneous repair after a stroke (Cheatwood et al., 2008). The blockade of NogoA promotes axonal growth (Domeniconi & Filbin, 2005), and this has been reported to be partially related to the behavioral recovery after a stroke (Lee et al., 2004; Zai et al., 2009; Zhao et al., 2013). NogoA interacts with NogoR to further active its downstream target Ras homolog gene family member A (RhoA)/Rho-associated kinase (ROCK) signal pathway, which could lead to growth cone collapse and axonal growth inhibition (Pernet et al., 2012). Therefore, it may be possible to enhance axonal growth and behavioral recovery after stroke by counteracting or reversing the inhibition of NogoA/NogoR and RhoA/ROCK (Lee et al.,2004).

Synapses are the fundamental structure of excitatory and inhibitory signal transmission in the central nervous system (CNS). The injury of synapses induced by stroke could lead to both sensorimotor and cognitive impairment (Hayakawa et al., 2010). Postsynaptic density-95 (PSD-95) is a key postsynaptic protein and vGlut-1 is an important presynaptic protein. The expression of these two proteins could represent the reactive synaptogenesis (Han & Kim, 2008; Seo et al., 2015; Tarsa & Goda, 2002).

Therefore, we investigated the effects of muscimol on axonal growth, synaptic plasticity and behavioral performance in rats after a focal ischemia induced by endothelin-1 (ET-1), as recommended by the Stroke Recovery and Rehabilitation Roundtable (SRRR) for recovery studies (Corbett et al., 2017). In addition, we used multiple behavioral tests to evaluate the treatment effects on sensorimotor functions of impaired limbs (Allred et al., 2005; Bouet et al., 2009; Hua et al., 2002).

2. Materials and methods

2.1. Animals

All the experimental procedures were approved by the Institutional Animal Care and Use Committee of China Medical University [SYXK [9] 2013-0007]. Wistar male rats were from Beijing Vital River Laboratory Animal Technology Co. Forty-seven rats were randomly divided into 3 groups: sham-operated rats (SHAM; n = 14), endothelin-1 induced focal ischemic rats (ISC; n = 16), ischemic rats treated with GABA_A receptors agonist muscimol (ISC+muscimol; n = 17). All experimental rats were housed in a temperature controlled environment with a 12-hour day-night circle. There was a maximum of five rats in each cage and food and water were freely available.

2.2. ET-1 stroke model

Anesthesia was induced by 4.5% isoflurane in compressed air (30% O2 and 70% N2). During the ischemic procedure, anesthesia was maintained with 1.5% to 2.5% isoflurane inhalation delivered via a nose mask. The following three sites were used for ET-1 microinjection (2 μl ET-1 (0.5 μg/μl) at a rate of 0.5 μl/min): (1) anteroposterior (AP) + 0.7 mm, mediolateral (ML) + 2.2 mm, dorsoventral (DV) –2.0 mm; (2) AP + 0.7 mm, ML + 3.8 mm, DV –5.8 mm; (3) AP + 2.3 mm, ML + 2.5 mm, DV –2.0 mm. The rectal temperature was monitored during the operative procedure. Sham-operated rats received the same volume of normal saline as with ET-1 injection.

2.3. In vivo drug administration

Muscimol (Tocris Bioscience, Bristol, UK) was dissolved in 0.9% saline to obtain a final concentration of 10 mM muscimol. Alzet 2002 (0.5 μl/h, 14-day, CA, USA) pumps containing 10 mM muscimol were activated overnight in a water bath (37°C) before implantation. The cannula (0.36 mm, Brain infusion kit 1; Alzet, CA, USA) connected to a pump was implanted into the contralateral cortex at a depth of 1.5 mm under the cortical surface (AP + 0.5 mm; ML + 0.3 mm) under isoflurane inhalation anesthesia (4.5% induction and 1.5% to 2.5% maintenance) (Hiu et al., 2016). The timing of drug administration is day 7 to 21 after stroke (Fig. 1A), which is according to the considerations mentioned in the discussion.

Fig.1

Flow chart of experimental design and Schematic map of ischemic infarct. A, Experimental design. The arrows show the time points of pre-training, endothelin-1(ET-1) injection, muscimol infusion, BDA injection, behavioral tests and euthanize respectively. B, Hand-drawing of ischemic infarcts.

2.4. Anterograde tracing of corticospinal tract (CST)

Biotinylated Dextran Amine (BDA, Invitrogen; 10% wt/vol solution in 0.01 mol/L PBS) was injected under isoflurane anesthesia on post-operative day 14 into the contralateral motor cortex to label CST fibers (Fig. 1A). The following coordinates were used for injections (2 μl 10% BDA per site, 0.5 μl/min): (1) AP + 1.0 mm, ML –2.0 mm, DV –2.0 mm/DV –1.5 mm; (2) AP 0 mm, ML –1.5 mm, DV –2.0 mm/DV –1.5 mm; (3) AP –1.0 mm, ML –1.4, DV –2.0 mm/DV –1.5 mm; (4) AP + 2.0 mm, ML –1.4 mm, DV –2.0 mm/DV –1.5 mm. After each injection, the needle was left in place for 3 minutes.

2.5. Postoperative care and observation

The operations that animals underwent in our study were made under the anesthesia of 1.5–2.5% isoflurane mixed with compressed air (30% O2 and 70% N2). All the operations were performed in the SPF animal laboratory and followed the principle of strict aseptic conditions. No rats were infected after the operation. After the surgery, the animals received a regimen of Buprenorphine (B-901, Sigma, USA) induced pain relief for 3 days (0.05 mg/kg, SC, twice daily) (Curtin et al., 2009; McKeon et al., 2011). All animals were looked after carefully by providing the suitable environment and enough food and water.

2.6. Behavioral analysis

Tapered/ledged beam-walking test was used to assess the motor functions of impaired (right) limbs at 32 days to 34 days after stroke onset. Rats were placed on the end of a tapered balance beam with two ledges on both sides. The rat’s performance was videotaped and the slip ratio for impaired limbs was calculated as follows: slip ratio = number of slips/number of total steps (Zhao et al., 2005).

Cylinder test was used to evaluate the spontaneous forelimb use on days 32 to 34 after the ischemic injury. Rats were placed in a transparent glass cylinder (23 cm diameter and 48 cm height) to monitor their exploration during 3 minutes. The whole procedure was recorded to count the number of times each forelimb was placed on the cylinder wall. The forelimb asymmetry score was estimated as follows: (contact with paralytic forelimb +1/2 contact with both forelimbs)/total number of all contacts (Allred et al., 2005).

Sticky label test is sensitive to detect sensorimotor function (Bouet et al., 2009). Small adhesive labels (4 mm×5 mm) were placed on both forepaws of a rat, which was returned to a test cage similar to its home cage. The time needed to contact the label and time needed for its removal were analyzed from a video recording. If the tapes could not be removed within 2 minutes, the rat was excluded.

2.7. Tissue preparation

On postoperative day 35, 9 rats (average 3 from each group) were deeply anesthetized with isoflurane and perfused with 0.9% normal saline for western blot analysis. The tissue specimens from the perilesional cortex (3 mm×3 mm, Fig. 1B) and C5–7 spinal cords were sampled. These samples were rapidly frozen in liquid nitrogen and stored at –80°C. The rest of the rats were perfused with 4% paraformaldehyde (PFA) after terminal anesthesia. The intact brain and C5–7 spinal cord was sampled into 4% PFA and stored at 4°C. After 24 hours’ post-fixation, the samples were dehydrated in gradient sucrose for 7 days in 4°C and tissues were embedded (Tissue-Tek, SAKURA Finetek, Tokyo, Japan). The brain tissue was cut (Thermo Electron, Waltham, MA, USA) into coronal sections (30 μm and 10 μm) for immunofluorescence staining. The spinal cord was cut into 50 μm sections for BDA staining and 30 μm sections for synaptic markers staining, respectively.

2.8. Immunohistochemical analysis

The sections were rinsed in 0.01M PBS and then incubated in the following primary antibodies at 4°C overnight after blocking in 5% –10% goat serum for 90 min: anti-Nogo A (1 : 400, Abcam), anti-Nogo Receptor (NogoR; 1 : 400, Abcam), anti-RhoA (1 : 500, Abcam), anti Rho-associate kinase (ROCK; 1 : 1000, Abcam), anti-postsynaptic density-95 (PSD-95; 1 : 800, Abcam) and anti-vGlut1 (1 : 100, Abacm). After washing for 3 times×10 min, Brain sections were incubated with conjugated streptavidin antibody (1 : 200, Alexa Flour 594, Invitrogen), Alexa Fluor 488 or 594 (1 : 200, Invitrogen) and spinal cord sections were incubated with conjugated streptavidin antibody (1 : 200, Alexa Flour 594, Invitrogen) or Alexa Flour 488 (1 : 200, Invitrogen) for 90 min.

2.9. Western blot analysis

Total protein in samples was extracted with a protein extraction kit (keyGEN BioTECH, China), and protein concentrations were measured by BCA protein concentration determination reagent kit (Beyotime, China). The primary antibodies were anti-Nogo A (1 : 1000, Abcam), anti-Nogo Receptor (NogoR; 1 : 300, Abcam), anti-RhoA (1 : 5000, Abcam), anti Rho-associate kinase (ROCK; 1 : 1000, Abcam), anti-postsynaptic density-95 (PSD-95; 1 : 350, Abcam) and anti-vGlut1 (1 : 1000, Abacm). The secondary antibody was goat anti-rabbit IgG H&L (HRP) (1 : 5000, Abcam). An ECL chemiluminescence system (Perice) was used to detect specific proteins.

2.10. Quantification

Six sections (50 μm thickness spinal cord section and 30 μm thickness brain section) of reconstruction of BDA-labeled axonal fibers in each rat were observed in a confocal microscope (Olympus FV-1000, Japan). The total length of midline crossing CST fibers was calculated at 10× magnification (brain section) and 20× magnification (spinal cord section) from three-dimensional reconstruction of z-series stacks with ImageJ (Figs. 2A, and 3A).

Fig.2

Effect of muscimol on biotinylated dextran amine (BDA) labeling crossing midline corticospinal tract (CST) in intrahemispheric stratum. A, Representative fluorescence images of BDA-immunolabeling of CST fibers (above: 10×; below: 40×). B, Total length of the CST axon crossing the midline. The data are present as the mean±S.D. ^{# #
#} P < 0.001 compared with SHAM group, Scale bar = 100 μm (10×); 50 μm (40×), N = 9.

Fig.3

Effect of muscimol on biotinylated dextran amine (BDA) labeling crossing midline corticospinal tract (CST) in C5–7 denervated spinal cord. A, Representative photographs of BDA labeling sprouting axons of CST from contralateral motor cortex to denervated spinal cord. B, The total length of sprouting axons crossing midline. The data are present as the mean±S.D. ^{# #} P < 0.01 compared with SHAM group, Scale bar = 100 μm, N = 11.

The expressions of synaptic markers (PSD-95, vGlut-1) were detected by both immunofluorescence and western blotting. Images of PSD-95/vGlut-1 (30 μm thickness) in each rat were captured using Olympus FV-1000 (Japan) at 20× magnification from the ventral horn. The integrated density of 6 images from each rat was measured by ImageJ. Western blot images were captured by ECL Chemiluminescence Apparatus and their gray value were detected by Image-Pro Plus 6.0.

To confirm the expression of growth inhibitors (NogoA, NogoR, RhoA, ROCK), they were also detected by immunofluorescence and western blot (Fig. 1B) as with the expression of synaptic markers. The number of immunopositive cells in the perilesional cortex was counted in 20× magnification immunofluorescence images by ImageJ.

2.11. Statistics

SPSS software 17 was used to analyze the data. All data were given as mean±SD. The one-way ANOVA were used to estimate statistical differences. P < 0.05 was considered as statistical significance. Power analysis was performed using previous data generated in our laboratory. For the total length of BDA labeled crossing midline corticospinal tract with a effect size of 0.749, a group size of 9 is required with alpha <0.05 and 90% power. For biochemistry experiments (IF) of axonal growth inhibitors with a effect size of 0.857, a group size of 9 is required to generate with alpha <0.05 and 90% power. The power of each testing index is mainly above 0.8 based on the calculation of power in the study.

2.12. Randomization and blinding

Stroke rats were randomly grouped into ischemic group and Muscimol treatment groups. The investigators did not know the sample group during the experiment and when assessing its outcome in this study.

3. Results

3.1. Survival of animals

Two ischemic rats out of 33 operated rats died. Three rats were excluded because they displayed difficulties in completing the behavioral tasks. All sham-operated rats survived.

3.2. Muscimol did not increase the growth of crossing CST after focal stroke

CST fibers sprouted collaterals to innervate the lesioned side stratum by crossing the midline after the cerebral ischemia. The length of the CST fibers crossing into the denervated side in the ISC group increased compared with the SHAM group (745.1±28.5 μm vs 249.1±18.5 μm, P < 0.001). However, muscimol treatment did not increase the number and length of the CST fibers crossing into the denervated stratum in comparison with the ISC group (772.4±19.8 μm vs 745.1±28.5 μm, P > 0.05) (Fig. 2AB).

In SHAM-operated rat spinal cord C5–7, there were only a few crossing midline axons of CST fibers (433.6±76.6 μm). On day 35 after the ischemic operation, the length of crossing midline fibers in the ISC group significantly increased (899.4±104.8 μm, P < 0.01 vs SHAM). In addition, there was no significant difference between the ISC group and the ISC + muscimol group (899.4±104.8 μm vs 806.1±179.6 μm, P > 0.05) (Fig. 3AB).

3.3. Muscimol did not affect the expression of axonal growth inhibitors after focal stroke

In order to explore the relationship between contralateral motor cortex inhibition and the behavioral recovery of ischemic rats, we analyzed the expression of various axonal growth inhibitors. There was no significant difference between ISC group and ISC+Muscimol group in the number of NogoA+/DAPI+ (Fig. 4), NogoR+/DAPI+ (Fig. 5), RhoA+/DAPI+ (Fig.6), and ROCK+/DAPI+ (Fig.7) positive cells in the perilesional cortex (Fig. 8A, ISC vs ISC + muscimol, P > 0.05). Western blotting confirmed that muscimol had not reduced the expression of the axonal growth inhibitors (Fig. 8BC, ISC vs ISC + muscimol, P > 0.05).

Fig.4

Representative fluorescence images of NogoA, green is NogoA labeling positive cells, blue is DAPI labeling positive cells, Scale bar = 100 μm, N = 11.

Fig.5

Representative fluorescence images of NogoR, red is NogoR labeling positive cells, blue is DAPI labeling positive cells, Scale bar = 100 μm, N = 11.

Fig.6

Representative fluorescence images of RhoA, red is RhoA labeling positive cells, blue is DAPI labeling positive cells, Scale bar = 100 μm, N = 11.

Fig.7

Representative fluorescence images of ROCK, green is ROCK labeling positive cells, blue is DAPI labeling positive cells, Scale bar = 100 μm, N = 11.

Fig.8

Effect of muscimol on NogoA/NogoR and RhoA/ROCK pathway in peri-infact cortex. A, Representative fluorescence images of NogoA, NogoR, RhoA, ROCK, blue is DAPI labeling positive cells and quantification data of these fluorescence double labeling positive cells respectively. B, Representative Western blots of NogoA, NogoR, RhoA and ROCK. C, Bar graphs of quantitative analysis protein expressions of Western blots. The data are present as the mean±S.D. ##P < 0.01 compared with SHAM group, Scale bar = 100 μ m, N = 11 (immunofluorescence); N = 3 (Western blot).

3.4. Partial restoration of synaptic markers by muscimol treatment

We applied both immunohistochemistry and western blotting to examine the effect of focal stroke on denervated spinal cord synapses by assessing the expression of vGlut-1 in presynaptic neurons and PSD-95 in postsynaptic neurons (Leal et al., 2014; Mestikawy et al., 2011). Muscimol significantly elevated the expression of PSD-95 (Fig. 9ABC; ISC vs ISC + muscimol, P < 0.01) but had no effect on vGlut-1 (Fig. 9ABC; ISC vs ISC + muscimol, P > 0.05).

Fig.9

Effect of muscimol on synaptic markers vGlut-1 and PSD-95. A, Representative fluorescence images of vGlut-1, PSD-95, and quantification data analysis of immunofluorescence. B, Representative Western blots of vGlut-1 and PSD-95. C, Bar graphs of quantitative analysis protein expressions of Western blots. The data are present as the mean±S.D. ##P < 0.01 compared with SHAM group, P < 0.05, Scale bar = 100μ m, N = 9 (immunofluorescence); N = 3 (Western blot).

Effect of muscimol on NogoA/NogoR and RhoA/ROCK pathway in peri-infact cortex. A, Representative fluorescence images of NogoA, NogoR, RhoA, ROCK, blue is DAPI labeling positive cells and quantification data of these fluorescence double labeling positive cells respectively. B, Representative Western blots of NogoA, NogoR, RhoA and ROCK. C, Bar graphs of quantitative analysis protein expressions of Western blots. The data are present as the mean±S.D. ^{# #} P < 0.01 compared with SHAM group, Scale bar = 100 μm, N = 11 (immunofluorescence); N = 3 (Western blot).

Effect of muscimol on synaptic markers vGlut-1 and PSD-95. A, Representative fluorescence images of vGlut-1, PSD-95, and quantification data analysis of immunofluorescence. B, Representative Western blots of vGlut-1 and PSD-95. C, Bar graphs of quantitative analysis protein expressions of Western blots. The data are present as the mean±S.D. ^{# #} P < 0.01 compared with SHAM group, ^*P < 0.05, Scale bar = 100 μm, N = 9 (immunofluorescence); N = 3 (Western blot).

3.5. Significantly improved partial behavioral performances by drug treatment

On post-operative day 32–34, behavioral outcomes were measured by tapered/ledged beam-walking test, cylinder test, and sticky label test.

In the tapered/ledged beam-walking test, there were significant increases in the paretic limbs slip ratio in the ISC group compared with normal controls (Fig. 10A, SHAM vs ISC, P < 0.01). The slip ratio of paretic limbs decreased after muscimol treatment (Fig. 10A, ISC vs ISC+ muscimol, P < 0.01).

Fig.10

Effect of muscimol on behavioral tests of ischemic rats. A, Bar graph of impaired forelimb and hindlimb (slip ratio) in beam-walking test, F represents for forelimb, H represents for hindlimb. B, Bar graph of limb use asymmetry in cylinder test. C, Bar graph of the time to touch the tape in sticky label test. D, Bar graph of the time to remove the tape in sticky label test. The data are present as the mean±S.D. ^{#
#} P < 0.01 compared with SHAM group, ^**P < 0.01 compared with ISC group, N = 14 (beam-walking test, sticky label test), N = 12 (cylinder test).

In the cylinder test, the asymmetry scores significantly increased in the ISC group compared with the SHAM group (Fig. 10B, SHAM vs ISC, P < 0.01). However, there was no significant difference between the ISC and ISC+muscimol groups (Fig. 10B, ISC vs ISC+muscimol, P > 0.05).

In the sticky label test, the time to contact the labels and time to remove them significantly increased after the stroke (Fig. 10CD, SHAM vs ISC, P < 0.01). After muscimol treatment, the time to contact the labels significantly decreased compared with ISC group (Fig. 10C, ISC vs ISC + muscimol, P < 0.01), but the time to remove labels did not differ from that of the ISC groups (Fig. 10D, ISC vs ISC + muscimol, P > 0.05).

4. Discussion

The present study investigated the effects of muscimol, a GABA_A Rs agonist on axonal growth, synaptic plasticity and behavioral recovery of focal stroke rats. The result demonstrated that muscimol did not affect the axonal sprouting from the contralateral CST to peri-infarct striatum and denervated spinal cord. Interestingly, muscimol treatment restored partially the behavioral performance.

The ET-1 model in this study does induce a cortical and striatal lesion. There are studies demonstrating that damage in the striatum may affect the hindlimb function and that this anatomical area is distinctively important for the sensorimotor control of the limbs (Biernaskie & Corbett, 2001; Langdon et al, 2011; Pisa, 1988). The behavioral defects of ischemic rats in our study is consistent with previous reports (Biernaskie & Corbett, 2001; Langdon et al., 2011; Pisa, 1988). It has been described that cortical lesions produce a maximal loss of projections from it into the dorsolateral striatum (McGeorge & Faull, 1989). This loss is then followed by massive sprouting of axons, from the contralateral cortex into the denervated striatum and the perilesion cortex (Carmichael & Chesselet, 2002; Dijkhuizen et al., 2003; Napieralski et al., 1996; Uryu et al., 2001). This axonal sprouting phenomenon could contribute to the functional recovery after brain injury (Overman et al., 2012; Riban & Chesselet, 2006). Muscimol delivery in our study produces a statistically significant improvement in behavioral recovery of motor and sensory function of paretic limbs after stroke. Though we did not detect axonal sprouting to the perilesion cortex, previous studies have shown that an increased axonal spouting in the perilesion cortex, after ischemic lesion (Carmichael & Chesselet, 2002). Furthermore, our study proves that muscimol treatment significantly increased axonal sprouting from the contralateral cortex into the perilesion striatum compared with the sham operated group. The striatum plays a critical role in motor and cognitive behaviors (Evart & Wise, 1984) and receives a massive excitatory input from the cerebral cortex (Somogyi et al., 1981). Therefore, we speculate that the muscimol treatment benefits the recovery from lesions in both the neocortex and striatum.

The timing (day 7 to 21 after stroke) of drug administration in our study is based on the following consideration. First, the repair phase of ischemic rats is about days to months after stroke (Hiu et al., 2016). Axonal sprouting after stroke progresses through specific biological time points. A trigger phase is present 1 to 3 days after stroke, in which rhythmic and synchronized neuronal discharges induce axonal sprouting (Carmichael & Chesselet, 2002). Seven and 14 days after the lesion are initiation and maintenance phases of the sprouting response in stroke and other CNS lesions (Leon et al., 2000; Steward, 1995; Stroemer et al., 1995). Secondly, both the molecular and cellular data support the idea that 2-to 3-week post-stroke interval is a window for axonal sprouting (Carmichael, 2006). On the other hand, the inactivation of the contralesional hemisphere induced by muscimol osmotic pumping for 14 days could get the best behavioral improvement after stroke (Mansoori et al., 2014). Besides, the literature shows that enhancing inhibition by muscimol immediately after stroke is benefit to the behavioral recovery of ischemic rats (Hiu et al., 2016); however, another study has shown that starting muscimol treatment at 3 day after stroke is harm to behavioral recovery (Lee et al., 2011). To minimize potential effects on acute injury size, we deliberately started muscimol treatment at 7 days after the onset of stroke. This allowed us study the effect of GABA_A receptors activation in brain plasticity. In general, taking day 7 to 21 after stroke as drug administration timing could better overlapping the axonal sprouting period after stroke.

Stroke could induce a degree of plasticity in structural and functional connections, including neurogenesis, axonal sprouting, angiogenesis and dendritic spine remodeling (Benowitz & Carmichael, 2010). It has been proposed that post-stroke axonal sprouting contains distinct phases-trigger, initiation, maintenance, and maturation (Benowitz & Carmichael, 2010; Carmichael, 2006). The emerging pathophysiological events in the trigger phase is the initial step of axonal sprouting after stroke. The trigger of axonal sprouting induces the expression of genes associated with this process and regulates its following phases in a complex manner (Carmichael, 2006). The increase in neuronal growth-promoting genes could support enhanced axonal sprouting. These include, but not limited to, SCG10, SCLIP, stathmin, and Rb3 (Suh et al., 2004; Mori & Morii, 2002; Golub & Pico, 2005). However, not all the genes that are activated are beneficial towards inducing axonal sprouting. Examples of these proteins include EphA4, Lingo-1 (part of the Nogo Recptror1), p75, and TROY (Giger et al., 2010; Li et al., 2010; Lemmens et al., 2013). Therefore, axonal growth can be stimulated by blocking axonal growth inhibitors or increasing neuronal growth programs after stroke (Li et al., 2010; Overman et al., 2012; Li et al., 2015; Wahl et al., 2014). As the post-stroke trigger phase is indispensable to the procedure of axonal sprouting, it is possible that there is a limited axonal sprouting response with the increased expression of growth inhibitors after stroke (Chang et al., 2016; Cheatwood et al., 2008; Li et al., 2018).

Interestingly, in contrast to our hypothesis that enhancing GABA inhibition would increase the axonal growth, it seemed that ischemic rats receiving a continuous infusion with a GABAA Rs agonist (muscimol) for 2 weeks were not different from the ISC group in terms of either axonal growth or the expression of NogoA/NogoR and RhoA/ROCK.

Previous evidence has suggested that treatment with a GABAA Rs agonist could accelerate the behavioral recovery after a cerebral ischemic injury (Hiu et al., 2016; Mansoori et al., 2014). However, another study indicated that the application of muscimol decreased axonal growth after a traumatic injury (Lee et al., 2011). Therefore, the failure to observe any such effect in the present study might be attributable to the combination of beneficial phasic inhibition and detrimental tonic inhibition on the axonal growth after stroke. In addition, previous studies have used different ischemic models, drug injection time points, dosages and durations, which might account for the conflicting results (Cui et al., 2008; Green et al., 2000; Mansoori et al., 2014; Schwitzguebel et al., 2016).

Whether muscimol has infused into contralateral cortex and diffused into the lesioned cortex could be reflected by the behavioral performance of bilateral limbs during the drug treatment. To explore the influence of muscimol on contralateral cortex, we detect the behavioral performance of healthy limbs (left) at 2 weeks after the drug withdrawal. The result is that were no significant differences between each group in both the beam-walking and sticky label tests. However, since we did not detect the behavioral performance during the drug treatment and all the data were collected 2 weeks after muscimol treatment in this study, we cannot say about more immediate effects of the drug. We speculate that the muscimol treatment could ameliorate the cortical and striatal lesion induced by stroke, based on the enhancing axonal sprouting in the infarcted cortex and subsequent improvement in behavioral performance. Besides, the obvious autonomous activity inhibition of bilateral limbs could be observed in our study during the drug treatment. This behavioral inhibition phenomenon has also been reported in the study by Mansoori and colleagues (Mansoori et al., 2014). It is also possible that axonal sprouting from the uninjured brain to the area around infarct could influence the local microenvironment around the area of damage by a series of signaling molecules and their action (Li et al., 2010). Example of these include chondroitin sulfate proteoglycans (Shen et al., 2010), bone morphogenic protein 7 (Winters et al., 2014) and Nogo Receptor 1 (Giger et al., 2010). Therefore, the results of our study support the possibility that muscimol, when injected into the contralateral (uninjured) brain crosses into ipsilateral (lesioned) brain. However, there is no direct evidence in our study, and we rely previous on previous research to show how muscimol is diffused and influences the infarcted side cerebral hemisphere. Behavioral performance testing and the expression of few proteins are inadequate in order to make a conclusion that muscimol has crossed into the lesioned brain. This question needs further and in-depth exploration in future studies.

The report of Clarkson and co-workers (Clarkson et al., 2010) is an all-round study examining the regulation of excessive GABAergic tonic inhibition after stroke. In this study, the effects of both short and long reduction of tonic inhibition, on behavioral recovery after stroke, was evaluated. At the same time, the function of reducing total GABAergic inhibition by PTX coupled with L655,708 treatment was examined. The result of the study could be summarized in that whilst L655,708 treatment generally benefited functional recovery after stroke, the combined PTX + L655,708 treatment is only advantageous in a short time window after stroke. Therefore, in their paper, they proposed that increasing cortical excitability by too much or reducing phasic inhibition both negatively impact function recovery after stroke. In our study, muscimol treatment restored partially the behavioral performance without enhancing axonal sprouting and reducing axonal growth inhibitors. Muscimol, as a potent agonist, targets both synaptic GABAA Rs (mediating phasic inhibition) and extra-synaptic GABAA Rs (mediating tonic inhibition) (Ebert et al., 1997; Stórustovu & Ebert, 2006). According to previous studies, we could speculate that both increasing phasic inhibition and reducing tonic inhibition are beneficial towards behavior recovery in rats after stroke (Clarkson et al., 2010). Thus, after increasing phasic and tonic inhibition by muscimol at the same time, it is reasonable have a less remarkable behavioral improvement and no significant difference in axonal sprouting activity, as presented in this study. Therefore, our data confirmed and supplemented the conclusion of Clarkson’s important work from the other side by enhancing GABAergic inhibition in chronic phase of stroke with 2 weeks muscimol treatment.

We should note that the limitation of this study is that the cortical neuron structure or integrity in the contralateral and ipsilateral regions have not been detected to find the possible reason of the behavioral improvement seen without effects on axonal growth or inhibition of growth inhibitors. The neuron apoptosis, integrity and neurogenesis will be detected in our future research to provide more reliable data to study the basic mechanism of behavioral recovery after muscimol administration in ischemic rats.

Footnotes

Acknowledgments

This study was supported by following funds: The Shenyang Foreign Science Exchange and Cooperation Technical Critical Special Project (No. 17-129-6-00, Chuansheng Zhao). The Program of the Distinguished Professor of Liaoning Province, Neurology (Chuansheng Zhao). National Natural Science Foundation of China (No. 81372104, Chuansheng Zhao). The Program of High-Level Innovation Team Training of China Medical University (No. 2017CXTD02, Chuansheng Zhao).

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