**Therapeutic Effects of Risperidone against Spinal Cord Injury in a Rat Model of Asphyxial Cardiac Arrest: A Focus on Body Temperature, Paraplegia, Motor Neuron Damage, and Neuroinflammation**

**Tae-Kyeong Lee 1,†, Jae-Chul Lee 2,†, Hyun-Jin Tae <sup>3</sup> , Hyung-Il Kim 4,5 , Myoung Cheol Shin <sup>5</sup> , Ji Hyeon Ahn 2,6 , Joon Ha Park <sup>7</sup> , Dae Won Kim <sup>8</sup> , Seongkweon Hong <sup>9</sup> , Soo Young Choi <sup>1</sup> , Jun Hwi Cho 5,\* and Moo-Ho Won 2,\***


**Abstract:** Cardiac arrest (CA) causes severe spinal cord injury and evokes spinal cord disorders including paraplegia. It has been reported that risperidone, an antipsychotic drug, effectively protects neuronal cell death from transient ischemia injury in gerbil brains. However, until now, studies on the effects of risperidone on spinal cord injury after asphyxial CA (ACA) and cardiopulmonary resuscitation (CPR) are not sufficient. Therefore, this study investigated the effect of risperidone on hind limb motor deficits and neuronal damage/death in the lumbar part of the spinal cord following ACA in rats. Mortality, severe motor deficits in the hind limbs, and the damage/death (loss) of motor neurons located in the anterior horn were observed two days after ACA/CPR. These symptoms were significantly alleviated by risperidone (an atypical antipsychotic) treatment after ACA. In vehicle-treated rats, the immunoreactivities of tumor necrosis factor-alpha (TNF-α) and interleukin 1-beta (IL-1β), as pro-inflammatory cytokines, were increased, and the immunoreactivities of IL-4 and IL-13, as anti-inflammatory cytokines, were reduced with time after ACA/CPR. In contrast, in risperidone-treated rats, the immunoreactivity of the pro-inflammatory cytokines was significantly decreased, and the anti-inflammatory cytokines were enhanced compared to vehicle-treated rats. In brief, risperidone treatment after ACA/CPR in rats significantly improved the survival rate and attenuated paralysis, the damage/death (loss) of motor neurons, and inflammation in the lumbar anterior horn. Thus, risperidone might be a therapeutic agent for paraplegia by attenuation of the damage/death (loss) of spinal motor neurons and neuroinflammation after ACA/CPR.

**Keywords:** whole-body ischemia; cardiopulmonary resuscitation; drug-induced hypothermia; spinal motor neuron; inflammation; paraplegia

**Citation:** Lee, T.-K.; Lee, J.-C.; Tae, H.-J.; Kim, H.-I.; Shin, M.C.; Ahn, J.H.; Park, J.H.; Kim, D.W.; Hong, S.; Choi, S.Y.; et al. Therapeutic Effects of Risperidone against Spinal Cord Injury in a Rat Model of Asphyxial Cardiac Arrest: A Focus on Body Temperature, Paraplegia, Motor Neuron Damage, and Neuroinflammation. *Vet. Sci.* **2021**, *8*, 230. https://doi.org/10.3390/ vetsci8100230

Academic Editor: Bartosz Kempisty

Received: 23 August 2021 Accepted: 8 October 2021 Published: 13 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

CA abruptly ceases blood circulation and oxygen delivery to the entire body, induces ischemia in the whole body, and develops high mortality after CA/CPR [1,2]. Studies on CA have concentrated on the improvement in the rate of the return of spontaneous circulation (ROSC) after CPR [3,4]. It has been reported that CA is one of the causes of severe spinal cord injuries including paraplegia, which negatively affects the quality of life in patients [5–7]. It is well known that motor neurons located in the ventral horn of the spinal cord are very vulnerable to ischemia-reperfusion injury [8–10]. However, the factors protecting or attenuating the damage of spinal motor neurons following ischemic insults have been insufficiently reported yet.

It is well accepted that body temperature influences the outcome of ischemic injury in patients after the ROSC [11–14]. To date, hypothermia has been applied to increase the ROSC in order to improve the survival rate of patients with CA. Data using experimental animals indicate that early cooling after the ROSC provides neurological recovery, but delayed hypothermia after ROSC limits these beneficial effects [15,16].

Risperidone (RIS), a benzoxazole derivative, has been widely used as a secondgeneration antipsychotic drug and selective monoaminergic antagonist containing high affinity for serotonin type 2 (5-HT2A) and dopamine type 2 (D2) receptors in the limbic system [17,18]. Studies in 2003 and 2004 reported that RIS induced hypothermia in patients with brain disorders, such as schizophrenia [19,20]. In a recent experimental study, RIS induced hypothermia in gerbils and effectively protected cells or neurons from ischemiareperfusion injury in the hippocampus by attenuating glial activation and maintaining antioxidants [21].

Neuroinflammation is a major pathophysiologic feature following brain ischemic insults [22,23]. The inflammatory cascade is induced a few hours after ischemic insults, and inflammation may last for a few days or weeks as a delayed tissue reaction to the damage [24,25]. The inflammatory response is controlled through the balance between pro- and anti-inflammatory cytokines, and this balance disappears after ischemia [26]. It is well accepted that pro-inflammatory cytokines promote inflammatory processes and the processes worsen following ischemia-reperfusion, but anti-inflammatory cytokines inhibit pro-inflammatory cytokine expression and induce ischemic tolerance [27,28].

There are some explanations of the protective effects of hypothermia against ischemic damage in the spinal cord [29,30], and we hypothesized that treatment with RIS after asphyxial CA (ACA) attenuates paraplegia and affects neuroinflammation in the spinal cord of patients with ACA. In this regard, we developed a rat model of ACA and examined the effects of RIS on paraplegia, neuronal damage and death, and inflammatory cytokines in the lumbar part of the spinal cord in rats following ACA/CPR.

#### **2. Materials and Methods**

#### *2.1. Rats, Protocol, and Groups for Experiment*

Male Sprague-Dawley rats at 10 weeks of age (body weight, 310–320 g) were obtained from the Experimental Animal Center of Kangwon National University (Chuncheon, Republic of Korea). The rats were kept under pathogen-free conditions with suitable temperature (about 23 ◦C) and humidity (about 60%). Freely accessible feed (DBL Co., Ltd.; Chungbuk, Korea) and water were provided to the rats. A 12-h cycle of light and dark was maintained.

The protocol for this experiment was approved on 18 February 2020 (approval no., KW-200113-1) by the Institutional Animal Care and Use Committee (IACUC). The protocol content adhered to the guidelines, which are in compliance with the "Current International Laws and Policies" from the "Guide for the Care and Use of Laboratory Animals" (The National Academies Press, 8th ed., 2011) [31]. The number of the rats used in this study was minimized, and the suffering caused by the procedures used in this experiment was minimized.

Rats (total *n* = 84) were assigned to four groups and treated as follows (Figure 1): (1) Sham+vehicle group (*n* = 21), which was given identical anesthetic and sham ACA/CPR operation, and intraperitoneally injected with vehicle; (2) ACA/CPR+vehicle group (*n* = 21 at each point in time), which was given ACA/CPR operation and intraperitoneally injected with vehicle; (3) Sham+RIS group (*n* = 21 at each point in time), which was given sham ACA/CPR operation and intraperitoneally injected with RIS; and (4) ACA/CPR+RIS group (*n* = 21), which was given ACA/CPR operation and injected intraperitoneally with RIS. In each group, seven rats were sacrificed at 12 h, one day and two days after ACA/CPR. ACA/CPR operation and intraperitoneally injected with RIS; and (4) ACA/CPR+RIS group (*n* = 21), which was given ACA/CPR operation and injected intraperitoneally with RIS. In each group, seven rats were sacrificed at 12 h, one day and two days after ACA/CPR. For reference, the original number of the rats used in this study was different (*n* = used number/original number) due to the survival rate as follows: (1) Sham+vehicle group (*n* = 7/7 at each time); (2) ACA/CPR+vehicle group (*n* = 7/9 at 12 h; *n* = 7/16 one day; *n* = 7/164 two days); (3) Sham+RIS group (*n* = 7/7 at each time); (4) ACA/CPR+RIS group (*n* = 7/8 at 12 h; *n* = 7/8 one day; *n* = 7/11 two days).

protocol content adhered to the guidelines, which are in compliance with the "Current International Laws and Policies" from the "Guide for the Care and Use of Laboratory Animals" (The National Academies Press, 8th Ed., 2011) [31]. The number of the rats used in this study was minimized, and the suffering caused by the procedures used in this exper-

Rats (total *n* = 84) were assigned to four groups and treated as follows (Figure 1): (1) Sham+vehicle group (*n* = 21), which was given identical anesthetic and sham ACA/CPR operation, and intraperitoneally injected with vehicle; (2) ACA/CPR+vehicle group (*n* = 21 at each point in time), which was given ACA/CPR operation and intraperitoneally injected with vehicle; (3) Sham+RIS group (*n* = 21 at each point in time), which was given sham

*Vet. Sci.* **2021**, *8*, x 3 of 15

iment was minimized.

**Figure 1.** Experimental timeline. The rats used in this study underwent sham or ACA/CPR followed by treatment with vehicle or 10 mg/kg RIS. They were deeply anesthetized and sacrificed at 12 h, 1 day, and 2 days after ROSC, and their spinal cords were used for analyses. **Figure 1.** Experimental timeline. The rats used in this study underwent sham or ACA/CPR followed by treatment with vehicle or 10 mg/kg RIS. They were deeply anesthetized and sacrificed at 12 h, 1 day, and 2 days after ROSC, and their spinal cords were used for analyses.

*2.2. ACA/CPR Operation and RIS Treatment* As shown in Figure 1, ACA/CPR was performed. Each rat was anesthetized with 2.5% isoflurane (Hana Pharmaceutical Co., Ltd.; Seoul, Korea) (in 33% oxygen and 67% nitrous oxide) and endotracheally intubated with a cannula (14-gauge) under mechanical ventilation with 2% isoflurane (in 33% oxygen and 67% nitrous oxide). Under the anes-For reference, the original number of the rats used in this study was different (*n* = used number/original number) due to the survival rate as follows: (1) Sham+vehicle group (*n* = 7/7 at each time); (2) ACA/CPR+vehicle group (*n* = 7/9 at 12 h; *n* = 7/16 one day; *n* = 7/164 two days); (3) Sham+RIS group (*n* = 7/7 at each time); (4) ACA/CPR+RIS group (*n* = 7/8 at 12 h; *n* = 7/8 one day; *n* = 7/11 two days).

#### thesia, the right femoral artery and vein were isolated and cannulated with catheters (PE-*2.2. ACA/CPR Operation and RIS Treatment*

50) to administer drug and to monitor arterial blood pressure. During the surgery of ACA/PCR, the body temperature in the rats was monitored using a rectal temperature probe (TR-100) (Fine Science Tools, Foster City, CA, USA) and maintained at a normothermic condition (37 ± 0.5 °C) using a thermometric blanket (Harvard Apparatus™, Holliston, MA, USA). Two mg/kg of vecuronium bromide obtained from Reyon Pharmaceutical (Seoul, Korea) was intravenously injected at 5 min after stabilization, and the anesthesia was stopped. Then, the mechanical ventilation in the rats was stopped, and the endotracheal tube was removed from the ventilator. Usually, ACA was confirmed at 3–4 As shown in Figure 1, ACA/CPR was performed. Each rat was anesthetized with 2.5% isoflurane (Hana Pharmaceutical Co., Ltd.; Seoul, Korea) (in 33% oxygen and 67% nitrous oxide) and endotracheally intubated with a cannula (14-gauge) under mechanical ventilation with 2% isoflurane (in 33% oxygen and 67% nitrous oxide). Under the anesthesia, the right femoral artery and vein were isolated and cannulated with catheters (PE-50) to administer drug and to monitor arterial blood pressure. During the surgery of ACA/PCR, the body temperature in the rats was monitored using a rectal temperature probe (TR-100) (Fine Science Tools, Foster City, CA, USA) and maintained at a normothermic condition (37 ± 0.5 ◦C) using a thermometric blanket (Harvard Apparatus™, Holliston, MA, USA). Two mg/kg of vecuronium bromide obtained from Reyon Pharmaceutical (Seoul, Korea) was intravenously injected at 5 min after stabilization, and the anesthesia was stopped. Then, the mechanical ventilation in the rats was stopped, and the endotracheal tube was removed from the ventilator. Usually, ACA was confirmed at 3–4 min after vecuronium bromide injection in this study. Perfect ACA was confirmed when pulseless electric activity (PEA) was shown and mean arterial pressure (MAP) was below 25 mmHg [8,9]. ACA was maintained for 5 min. Then, CPR was immediately initiated by an intravenous injection of 0.005 mg/kg of epinephrine (Dai Han Pharm, Seoul, Korea) and 1 meq/kg of sodium bicarbonate (Daewon Pharm, Seoul, Korea), and mechanical ventilation with

100% oxygen was simultaneously given. Subsequently, manual chest compressions were performed. Namely, manual chest compression was performed at a rate of 300/min until MAP increased to 60 mmHg, and electrocardiography was checked [8,9]. Once each rat breathed and was hemodynamically stable, which was usually shown 1 h after ROSC, the catheter was removed. The rat came out from the anesthesia 1 h after ROSC. For the control of body temperature from 20 min to 6 h after ACA, any artificial maintenance for body temperature was not conducted after ROSC while the ambient temperature (room temperature) was kept at 24 ± 1 ◦C.

In this study, the rats of the sham group underwent the surgical procedure of ACA without the injection of vecuronium. After the surgical procedure, the rats were placed in cages (DBL Co., Ltd.; Chungbuk, Korea), in which aspen beds were spread on the bottom, and they were kept in thermal incubators (Mirae Medical Industry, Seoul, Korea) at 25 ◦C and 60% humidity. While the rats were kept in the incubators, room temperature was maintained at 24 ± 1 ◦C. Body temperature and MAP was recorded every minute from 0 to 20 min. Thereafter, till 1 h after ACA induction, body temperature and MAP was measured every 5 min. Especially, body temperature was recorded every 15 min from 1 to 6 h after ACA induction.

As shown in Figure 1, vehicle or RIS (10 mg/kg) (Sigma-Aldrich, St. Louis, MO, USA) was injected into the peritoneal cavity immediately after ACA/CPR operation. The dose of RIS was selected based on a previous study [21]. RIS was dissolved in 0.3% Tween 80 (in 0.85% saline; NaCl *w*/*v*; Junsei Chemical Co., Ltd., Tokyo, Japan).

#### *2.3. Assessment of Physiological Variables and Motor Function*

Body weight and MAP between the groups were compared at 1 day after ROSC. Motor function of the hind limbs was evaluated for paralysis at 1 day after ROSC using Tarlov Scale [8]: motor deficit scoring 0, complete paralysis with no hind limb function; 1, slight movement in articulations; 2, unable to stand without support; 3, sit alone; 4, weak walking with poor jumping; 5, normal walking.

#### *2.4. Preparation of Histological Sections*

The rats (*n* = 7 at each point in time) in each group were used for histopathological staining and immunohistochemistry at 12 h, 1 day, and 2 days after ROSC. The rats were deeply anesthetized by intraperitoneal injection of 200 mg/kg pentobarbital sodium (JW pharm Co Ltd., Seoul, Korea) [32]. Under the anesthesia, they were transcardially rinsed with 0.1 M phosphate-buffered saline (PBS, pH 7.4) and fixed with 4% paraformaldehyde (in 0.1 M PB, pH 7.4) for 30 min. The lumbar parts of the spinal cords were obtained and postfixed in the same fixative for 8 h. The lumbar spinal cords were infiltrated with 25% sucrose (in 0.1 M PB) to be cryoprotected for 12 h. To prepare histological sections, the spinal cord tissues were frozen in a cryostat (Leica, Wetzlar, Germany) and serially cut into a 25-µm coronal plane.

## *2.5. Fluoro-Jade B (F-J B) Histofluorescence*

F-J B (a fluorescent marker for cellular degeneration) histofluorescence was performed to assess neuronal damage/death (loss) after ACA/CPR. In short, as described previously [33], the spinal cord sections were immersed in 0.0004% F-J B (Histochem, Jefferson, AR, USA) and washed. Finally, for the reaction of the F-J B, these sections were placed on a slide warmer (about 50 ◦C).

To quantitatively analyze the death or protection of motor neurons in the ventral horn, five sections were chosen with a 120-µm interval. F-J B-positive cells were counted as previously described [34]. In short, F-J B-positive cells (neurons) were observed with an epifluorescence microscope (BX53) (Olympus, Tokyo, Japan) with blue (450–490 nm) excitation light. The images were captured with a digital camera (DP7) (Olympus, Tokyo, Japan) connected to a PC monitor. The F-J B-positive cells were counted in 200,000 µm<sup>2</sup> (400 µm × 500 µm) at the anterior horn. Counts of the cells were evaluated by averaging

the total numbers obtained from 35 sections from 7 rats/group using an image analyzing system (Optimas 6.5) from CyberMetrics (Scottsdale, AZ, USA).

#### *2.6. Immunohistochemistry*

In this study, general immunohistochemistry was carried out to examine changes regarding the neurons, pro-inflammatory, and anti-inflammatory cytokines. For the immunohistochemistry, we used primary antibodies as follows: mouse anti-neuronal nuclei (NeuN; diluted 1:1100; Cat. No., MAB377; Chemicon International, Temecula, CA, USA), rabbit anti-TNF-α (diluted 1:1200) (Cat. No., ab66579; Abcam, Cambridge, UK), rabbit anti-IL-1β (diluted 1:250) (Cat. No., ab2105; Abcam, Cambridge, UK), goat anti-IL-4 (diluted 1:200) (Cat. No. sc-1260; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and goat anti-IL-13 (diluted 1:200) (Cat. No., sc-393365; Santa Cruz Biotechnology, Santa Cruz, CA, USA). In short, as described previously [35], the sections were incubated with each diluted antibody for 12 h at 4 ◦C. After the sections were washed, they were reacted with biotinylated horse anti-mouse (diluted 1:200) (Cat. No., BA-2001;Vector Laboratories, Burlingame, CA, USA), goat rabbit (diluted 1:200) (Cat. No., BA-1000; Vector Laboratories, Burlingame, CA, USA), or rabbit anti-goat IgG (diluted 1:200) (Cat. No., BA-5000; Vector Laboratories, Burlingame, CA, USA) and, thereafter, developed by avidin-biotin complex (ABC) (diluted 1:300) (Cat. No. PK-4000; Vector Laboratories, Burlingame, CA, USA). Finally, they were visualized with 3,30 -diaminobenzidine solution (DAB; Cat. No., D8001; Sigma-Aldrich, St. Louis, MO, USA). The sections were identically reacted with DAB solution for 90 s at room temperature. In addition, negative control tests for NeuN, TNF-α, IL-1 β, IL-4, and IL-13 were performed for the specificity of each immunostaining, with pre-immune serum instead of each primary antibody. As a result, any immunostained structures were not shown in the tested sections.

For quantitative analysis of the number of NeuN immunoreactive motor neurons and their change, five sections/rat were chosen with a 120-µm interval. The numbers were counted as described in the Section 2.5.

For quantitative analysis of each immunoreactivity (TNF-α, IL-1 β, IL-4, and IL-13) in the ventral horn, the images were taken using the above-mentioned method and analyzed as described in our published paper [35]. Briefly, each image of the captured immunoreactivity was evaluated as optical density (OD): the OD was obtained after transforming each immunoreactive structure to mean gray level using the formula OD = log (256/mean gray level). Finally, each OD was compared as the relative optical density (ROD): a ratio of the ROD was evaluated as percent using Image J software (version 1.59) from NIH (Bethesda, MD, USA).

#### *2.7. Statistical Analysis*

In this study, SPSS software (version 15.0) from SPSS Inc (Chicago, IL, USA) was used to perform all statistical analysis. We used the Kolmogorov and Smirnov test for testing normal distributions and Bartlett test for testing the identical standard error of the means (SEMs), and all our data passed the normality test. The statistical significances of the mean among the experimental groups were determined by one-way analysis of variance followed by post hoc Tukey test for all pairwise multiple comparisons. Any differences lower than 0.05 of *p* value were considered significant.

#### **3. Results**

#### *3.1. Changes in Physiological Function and Body Temperature*

MAP and body temperature was recorded in each group before and after ACA operation as shown in Figure 2. Before ACA, MAP and body temperature were similar to the baselines observed in the Sham+vehicle group. Body temperature in the ACA/CPR+RIS group was not significantly different from that in the ACA/CPR+vehicle group (Figure 2A). Under 24 ± 1 ◦C of room temperature, a significant low body (rectal) temperature (33 ± 0.5 ◦C) in all RIS groups was detected from 1 to 2 h after ACA, which was due to RIS injection.

*Vet. Sci.* **2021**, *8*, x 6 of 15

than 0.05 of *p* value were considered significant.

*3.1. Changes in Physiological Function and Body Temperature*

*2.7. Statistical Analysis*

**3. Results**

Thereafter, their body temperature was spontaneously and gradually increased with intermittently shivering to 37 ± 0.5 ◦C (Figure 2B). injection. Thereafter, their body temperature was spontaneously and gradually increased with intermittently shivering to 37 ± 0.5 °C (Figure 2B).

In this study, SPSS software (version 15.0) from SPSS Inc (Chicago, IL, USA) was used to perform all statistical analysis. We used the Kolmogorov and Smirnov test for testing normal distributions and Bartlett test for testing the identical standard error of the means (SEMs), and all our data passed the normality test. The statistical significances of the mean among the experimental groups were determined by one-way analysis of variance followed by post hoc Tukey test for all pairwise multiple comparisons. Any differences lower

MAP and body temperature was recorded in each group before and after ACA operation as shown in Figure 2. Before ACA, MAP and body temperature were similar to the baselines observed in the Sham+vehicle group. Body temperature in the ACA/CPR+RIS group was not significantly different from that in the ACA/CPR+vehicle group (Figure 2A). Under 24 ± 1 °C of room temperature, a significant low body (rectal) temperature (33 ± 0.5 °C) in all RIS groups was detected from 1 to 2 h after ACA, which was due to RIS

**Figure 2.** MAP (**A**) and body temperature (**B**) before, during, and after ACA in the Sham+vehicle, ACA/CPR+vehicle, and ACA/CPR+RIS groups. Note that body temperature in the ACA/CPR+RIS group was 33 ± 0.5 °C from 1 to 2 h after ACA. a, inducing ACA; b, maintaining ACA condition; c, conducting CPR; d, confirming ROSC. The bars indicate the means ± SEM (*n* = 7). **Figure 2.** MAP (**A**) and body temperature (**B**) before, during, and after ACA in the Sham+vehicle, ACA/CPR+vehicle, and ACA/CPR+RIS groups. Note that body temperature in the ACA/CPR+RIS group was 33 ± 0.5 ◦C from 1 to 2 h after ACA. a, inducing ACA; b, maintaining ACA condition; c, conducting CPR; d, confirming ROSC. The bars indicate the means ± SEM (*n* = 7).

#### *3.2. Survival Rate and Motor Deficit Score 3.2. Survival Rate and Motor Deficit Score*

The survival rate in the ACA/CPR+vehicle and ACA/CPR+vehicle groups was recorded by Kaplan-Meier analysis for 2 days after ACA/CPR (Figure 3A). In all sham groups, all rats survived (Figure 3A). In the ACA/CPR+vehicle group, the survival rate gradually reduced with time after ACA/CPR, showing 65.3% at 1 day and 4.3% at 2 days after ROSC (Figure 3A). In the ACA/CPR+RIS group, however, the survival rate was The survival rate in the ACA/CPR+vehicle and ACA/CPR+vehicle groups was recorded by Kaplan-Meier analysis for 2 days after ACA/CPR (Figure 3A). In all sham groups, all rats survived (Figure 3A). In the ACA/CPR+vehicle group, the survival rate gradually reduced with time after ACA/CPR, showing 65.3% at 1 day and 4.3% at 2 days after ROSC (Figure 3A). In the ACA/CPR+RIS group, however, the survival rate was significantly high compared with that in the ACA/CPR+vehicle group, showing 92.4% at 1 day and 67.9% at 2 days after ACA/CPR (Figure 3A).

Hind limb motor deficit (paralysis) was evaluated with the Tarlov score at 1 day after ACA/CPR (Figure 3B). The rats of the Sham+vehicle group revealed normal function in their hind limbs. In the ACA/CPR+vehicle group, the score was significantly low (average 0.8 point) compared with that in the Sham+vehicle group (average 4.1 point) (*p* < 0.01). In the ACA/CPR+RIS group, however, motor function was significantly better (average 2.9 point) than that in the ACA/CPR+vehicle group (*p* < 0.05).

#### *3.3. Neuroprotection by RIS*

#### 3.3.1. NeuN Immunoreactive Neurons

We examined neuronal damage/loss in the ventral horn of the lumbar part in the spinal cord after ACA/CPR using immunohistochemistry with NeuN: NeuN is well used to detect neuronal nucleus damage (Figure 4). In the Sham+vehicle and Sham+RIS groups, neurons in the anterior horn, which are called motor neurons, were well stained with NeuN in their nuclei (Figure 4A(a,b,e,f)). In the ACA/CPR+vehicle group, a few neurons stained with NeuN (NeuN<sup>+</sup> neurons) were shown in the anterior horn at 2 days after ACA/CPR

(Figure 4A(c,g)). The mean percentage of NeuN<sup>+</sup> neurons, in this group, was 24.6% of that in the Sham+vehicle group (Figure 4C). However, in the ACA/CPR+RIS group, many NeuN<sup>+</sup> neurons were found at 2 days after ACA/CPR (Figure 4A(d,h)), revealing that the mean percentage of the motor neurons was 91.7% of that in the Sham+vehicle group (Figure 4C). ACA/CPR (Figure 3B). The rats of the Sham+vehicle group revealed normal function in their hind limbs. In the ACA/CPR+vehicle group, the score was significantly low (average 0.8 point) compared with that in the Sham+vehicle group (average 4.1 point) (*p* < 0.01). In the ACA/CPR+RIS group, however, motor function was significantly better (average 2.9 point) than that in the ACA/CPR+vehicle group (*p* < 0.05).

Hind limb motor deficit (paralysis) was evaluated with the Tarlov score at 1 day after

significantly high compared with that in the ACA/CPR+vehicle group, showing 92.4% at

*Vet. Sci.* **2021**, *8*, x 7 of 15

1 day and 67.9% at 2 days after ACA/CPR (Figure 3A).

**Figure 3.** Kaplan-Meier survival curve and Tarlov score (**A**) Survival rate (*p* < 0.05) in the Sham+vehicle, Sham+IRS, ACA/CPR+vehicle, and ACA/CPR+RIS groups using Kaplan–Meier analysis for 2 days after ACA/CPR. The ACA/CPR+RIS group reveals a higher survival rate than the ACA/CPR+vehicle group. At 2 days after ACA/CPR, the cumulative survival rate in the ACA/CPR+vehicle group is 4.3% whereas the cumulate survival rate in the ACA/CPR+RIP group is 67.9%. (**B**) Motor function of both hind limbs in the Sham+vehicle, Sham+RIS, ACA/CPR+vehicle, and ACA/CPR+RIS groups using Tarlov Scoring System. At 1 day after ACA, a significant higher score in the ACA/CPR+RIP group is observed compared to that in the ACA/CPR+vehicle group. The bars indicate the means ± SEM (*n* = 7; \* *p* < 0.05 vs. Sham+vehicle group; † *p* < 0.05 vs. ACA/CPR+vehicle group). **Figure 3.** Kaplan-Meier survival curve and Tarlov score (**A**) Survival rate (*p* < 0.05) in the Sham+vehicle, Sham+IRS, ACA/CPR+vehicle, and ACA/CPR+RIS groups using Kaplan–Meier analysis for 2 days after ACA/CPR. The ACA/CPR+RIS group reveals a higher survival rate than the ACA/CPR+vehicle group. At 2 days after ACA/CPR, the cumulative survival rate in the ACA/CPR+vehicle group is 4.3% whereas the cumulate survival rate in the ACA/CPR+RIP group is 67.9%. (**B**) Motor function of both hind limbs in the Sham+vehicle, Sham+RIS, ACA/CPR+vehicle, and ACA/CPR+RIS groups using Tarlov Scoring System. At 1 day after ACA, a significant higher score in the ACA/CPR+RIP group is observed compared to that in the ACA/CPR+vehicle group. The bars indicate the means ± SEM (*n* = 7; \* *p* < 0.05 vs. Sham+vehicle group; † *p* < 0.05 vs. ACA/CPR+vehicle group). *Vet. Sci.* **2021**, *8*, x 8 of 15

the anterior horn at 2 days after ACA/CPR (Figure 4Bc,D). In the ACA/CPR+RIS group, the numbers of F-J B<sup>+</sup> cells were significantly decreased at 2 days after ACA/CPR (Figure 4Bd), showing that the mean percentage of the F-J B<sup>+</sup> cells was 12.1% of that in the ACA/CPR+vehicle group (Figure 4D). **Figure 4.** NeuN immunohistochemistry and F-J B histofluorescence (**A**) NeuN immunohistochemistry in the lumbar spinal cord of the Sham+vehicle (**a**,**e**), Sham+RIS (**b**,**f**), ACA/CPR+vehicle (**c**,**g**), and ACA/CPR+RIS (**d**,**h**) groups at 2 days after ACA/CPR. The middle panels are high magnified images for the squares in the upper panels. In the ACA/CPR+vehicle group, NeuN+ neurons are rarely shown (asterisk) in the ventral horn (VH). However, many NeuN+ cells are shown in the ACA/CPR+RIS group. DH, dorsal horn. Scale bar = 200 (**a**–**d**) and 100 (**e**–**h**) µm. (**B**) F-J B histofluorescence in the ventral horn of the Sham+vehicle (**a**), Sham+RIS (**b**), ACA/CPR+vehicle (**c**), and ACA/CPR+RIS (**d**) groups at 2 days after ACA/CPR. In the ACA/CPR+vehicle group, many F-J B+ cells (arrows) are shown, but the numbers of F-J B+ cells are decreased in the ACA/CPR+RIS group. Scale bar = 400 µm (**A**) and 100 µm. (**C**,**D**) Quantitative analyses of NeuN+ (**C**) and F-J B+ cells (**D**) in the VH. The bars indicate the means ± SEM (*n* = 7; \* *p* < 0.05 vs. Sham+vehicle group; † *p* < 0.05 vs. ACA/CPR+vehicle group). *3.4. Decreased Pro-Inflammatory Cytokines by RIS* **Figure 4.** NeuN immunohistochemistry and F-J B histofluorescence (**A**) NeuN immunohistochemistry in the lumbar spinal cord of the Sham+vehicle (**a**,**e**), Sham+RIS (**b**,**f**), ACA/CPR+vehicle (**c**,**g**), and ACA/CPR+RIS (**d**,**h**) groups at 2 days after ACA/CPR. The middle panels are high magnified images for the squares in the upper panels. In the ACA/CPR+vehicle group, NeuN+ neurons are rarely shown (asterisk) in the ventral horn (VH). However, many NeuN+ cells are shown in the ACA/CPR+RIS group. DH, dorsal horn. Scale bar = 200 (**a**–**d**) and 100 (**e**–**h**) µm. (**B**) F-J B histofluorescence in the ventral horn of the Sham+vehicle (**a**), Sham+RIS (**b**), ACA/CPR+vehicle (**c**), and ACA/CPR+RIS (**d**) groups at 2 days after ACA/CPR. In the ACA/CPR+vehicle group, many F-J B+ cells (arrows) are shown, but the numbers of F-J B+ cells are decreased in the ACA/CPR+RIS group. Scale bar = 400 µm (**A**) and 100 µm. (**C**,**D**) Quantitative analyses of NeuN+ (**C**) and F-J B+ cells (**D**) in the VH. The bars indicate the means <sup>±</sup> SEM (*<sup>n</sup>* = 7; \* *<sup>p</sup>* < 0.05 vs. Sham+vehicle group; † *<sup>p</sup>* < 0.05 vs. ACA/CPR+vehicle group).

α immunoreactivity was gradually enhanced until 1 day after ACA, showing that the ROD of TNF-α immunoreactivity at 12 h and 1 day after ACA/CPR was 183.5% and 211.9%, respectively, compared with that in the Sham+vehicle group (Figure 5A(b,c),B). Thereafter, TNF-α immunoreactivity was decreased, but the ROD was 150.1% of that in

In the Sham+RIS group, TNF-α immunoreactivity in the ventral horn was similar to that in the Sham+vehicle group (Figure 5Ae,B). In addition, in the ACA/CPR+RIS group, TNF-α immunoreactivity in the anterior horn showed no difference from that in the

In the Sham+vehicle group, IL-1β immunoreactivity was weakly shown in the motor neurons (Figure 5Ca). In the ACA/CPR+vehicle group, IL-1β immunoreactivity at 12 h, 1 day, and 2 days after ACA/CPR was intensely increased, showing that the ROD was 224.4%, 305.3, and 237.1%, respectively, compared with that in the Sham+vehicle group

In the Sham+RIS group, IL-1β immunoreactivity in the lumbar ventral horn was not significantly different from that found in the Sham+vehicle group (Figure 5Be,D). In the ACA/CPR+RIS group, IL-1β immunoreactivity was gradually enhanced after ACA, but

3.4.1. TNF-α Immunoreactivity

the Sham+vehicle group (Figure 5Ad,B).

Sham+vehicle group (Figure 5A(f–h),B).

3.4.2. IL-1 β Immunoreactivity

(Figure 5B(b–d),C).

#### 3.3.2. F-J B-Positive Cells

The neuroprotection by RIS from ACA/CPR in the ventral horn was analyzed by F-J B histofluorescence: F-J B is an excellent marker for detection of dead cells (neurons) (Figure 4B). No F-J B-positive (F-J B<sup>+</sup> ) cells were found in the Sham+vehicle and Sham+RIS groups (Figure 4B(a,b)). In the ACA/CPR+vehicle group, many F-J B<sup>+</sup> cells were found in the anterior horn at 2 days after ACA/CPR (Figure 4Bc,D). In the ACA/CPR+RIS group, the numbers of F-J B<sup>+</sup> cells were significantly decreased at 2 days after ACA/CPR (Figure 4Bd), showing that the mean percentage of the F-J B<sup>+</sup> cells was 12.1% of that in the ACA/CPR+vehicle group (Figure 4D).

#### *3.4. Decreased Pro-Inflammatory Cytokines by RIS*

#### 3.4.1. TNF-α Immunoreactivity

TNF-α immunoreactivity shown in the Sham+vehicle group was shown in the motor neurons located in the anterior horn (Figure 5Aa). In the ACA/CPR+vehicle group, TNFα immunoreactivity was gradually enhanced until 1 day after ACA, showing that the ROD of TNF-α immunoreactivity at 12 h and 1 day after ACA/CPR was 183.5% and 211.9%, respectively, compared with that in the Sham+vehicle group (Figure 5A(b,c),B). Thereafter, TNF-α immunoreactivity was decreased, but the ROD was 150.1% of that in the Sham+vehicle group (Figure 5Ad,B). *Vet. Sci.* **2021**, *8*, x 9 of 15 the ROD at each point in time was significantly lower (41.2%, 45.4%, and 23.5%, respectively) than that in the ACA/CPR+vehicle group (Figure 5D).

**Figure 5.** Immunohistochemical staining for TNF-α and IL-1β (**A**,**C**) Immunohistochemistry for TNF-α (**A**) and IL-1β (**C**) in the ventral horn of the Sham+vehicle (**a**), ACA/CPR+vehicle (**b**–**d**), Sham+RIS (**e**), and ACA/CPR+RIS (**f**–**h**) groups at 12 h, 1 day, and 2 days after ACA/CPR. In the ACA/CPR+vehicle group, TNF-α and IL-1β immunoreactivities are significantly increased from 12 h after ACA/CPR. However, in the ACA/CPR+RIS group, immunoreactivities of TNF-α and IL-1β are significantly low compared with that shown in the ACA/CPR+vehicle group. VH, ventral horn. Scale bar = 100 µm. (**A**,**C**) RODs of TNF-α (**B**) and IL-1β (**D**) immunoreactivity. The bars indicate the means ± SEM (*n* = 7; \**p* < 0.05 vs. Sham+vehicle group; † *p* < 0.05 vs. ACA/CPR+vehicle group; # *p* < 0.05 vs. Pre-time point of corresponding group). **Figure 5.** Immunohistochemical staining for TNF-α and IL-1β (**A**,**C**) Immunohistochemistry for TNF-α (**A**) and IL-1β (**C**) in the ventral horn of the Sham+vehicle (**a**), ACA/CPR+vehicle (**b**–**d**), Sham+RIS (**e**), and ACA/CPR+RIS (**f**–**h**) groups at 12 h, 1 day, and 2 days after ACA/CPR. In the ACA/CPR+vehicle group, TNF-α and IL-1β immunoreactivities are significantly increased from 12 h after ACA/CPR. However, in the ACA/CPR+RIS group, immunoreactivities of TNF-α and IL-1β are significantly low compared with that shown in the ACA/CPR+vehicle group. VH, ventral horn. Scale bar = 100 µm. (**A**,**C**) RODs of TNF-α (**B**) and IL-1β (**D**) immunoreactivity. The bars indicate the means ± SEM (*n* = 7; \* *p* < 0.05 vs. Sham+vehicle group; † *p* < 0.05 vs. ACA/CPR+vehicle group; # *p* < 0.05 vs. Pre-time point of corresponding group).

*3.5. Increased Anti-Inflammatory Cytokines by RIS* 3.5.1. IL-4 Immunoreactivity IL-4 immunoreactivity in the ventral horn of the Sham+vehicle group was shown in the motor neurons (Figure 6Aa). In the ACA/CPR+vehicle group, IL-4 immunoreactivity was dramatically and gradually decreased after ACA/CPR, showing that the ROD at 12 In the Sham+RIS group, TNF-α immunoreactivity in the ventral horn was similar to that in the Sham+vehicle group (Figure 5Ae,B). In addition, in the ACA/CPR+RIS group, TNF-α immunoreactivity in the anterior horn showed no difference from that in the Sham+vehicle group (Figure 5A(f–h),B).

#### h, 1 day, and 2 days after ACA/CPR was 68.3%, 47.1%, and 31.3%, respectively, compared 3.4.2. IL-1 β Immunoreactivity

Sham+vehicle group (Figure 6B(b–d),D).

h),B).

6C(f–h),D).

with that found in the Sham+vehicle group (Figure 6A(b–d),B). In the Sham+RIS group, IL-4 immunoreactivity in the lumbar ventral horn was similar to that shown in the Sham+vehicle group (Figure 6Ae,B). In the ACA/CPR+RIS group, In the Sham+vehicle group, IL-1β immunoreactivity was weakly shown in the motor neurons (Figure 5Ca). In the ACA/CPR+vehicle group, IL-1β immunoreactivity at 12 h, 1 day, and 2 days after ACA/CPR was intensely increased, showing that the ROD was

IL-4 immunoreactivity in the anterior horn was maintained after ACA/CPR (Figure 6A(f–

In the ventral horn of the Sham+vehicle group, IL-13 immunoreactivity was also found in the motor neurons (Figure 6Ba). IL-13 immunoreactivity in the ACA/CPR+vehicle group was dramatically and gradually decreased after ACA/CPR (RODs: 81.7% at 12 h, 60.9% at 1 day, and 34.5% at 2 days after ACA/CPR) compared with that in the

In the Sham+RIS group, IL-13 immunoreactivity in the ventral horn was not different from that shown in the Sham+vehicle group (Figure 6Ce,D). In the ACA/CPR+RIS group, IL-13 immunoreactivity in the anterior horn was also maintained after ACA/CPR (Figure

224.4%, 305.3, and 237.1%, respectively, compared with that in the Sham+vehicle group (Figure 5B(b–d),C).

In the Sham+RIS group, IL-1β immunoreactivity in the lumbar ventral horn was not significantly different from that found in the Sham+vehicle group (Figure 5Be,D). In the ACA/CPR+RIS group, IL-1β immunoreactivity was gradually enhanced after ACA, but the ROD at each point in time was significantly lower (41.2%, 45.4%, and 23.5%, respectively) than that in the ACA/CPR+vehicle group (Figure 5D).
