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 h, 1 day, and 2 days after ACA/CPR was 68.3%, 47.1%, and 31.3%, respectively, compared with that found in the Sham+vehicle group (Figure 6A(b–d),B). *Vet. Sci.* **2021**, *8*, x 10 of 15

**Figure 6.** Immunohistochemical staining for IL-4 and IL-13 (**A**,**C**) Immunohistochemistry for IL-4 (**A**) and IL-13 (**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, immunoreactivities of IL-4 and IL-13 are significantly decreased from 12 h after ACA/CPR. However, in the ACA/CPR+RIS group, immunoreactivities of IL-4 and IL-13 are maintained after ACA/CPR. VH, ventral horn. Scale bar = 100 µm. (**A**,**C**) RODs of IL-4 (**B**) and IL-13 (**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 the corresponding group). **Figure 6.** Immunohistochemical staining for IL-4 and IL-13 (**A**,**C**) Immunohistochemistry for IL-4 (**A**) and IL-13 (**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, immunoreactivities of IL-4 and IL-13 are significantly decreased from 12 h after ACA/CPR. However, in the ACA/CPR+RIS group, immunoreactivities of IL-4 and IL-13 are maintained after ACA/CPR. VH, ventral horn. Scale bar = 100 µm. (**A**,**C**) RODs of IL-4 (**B**) and IL-13 (**D**) immunoreactivity. 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; # *p* < 0.05 vs. Pre-time point of the corresponding group).

> **4. Discussion** Generally, the spinal cord is prominently susceptible to ischemic insults owing to diverse circulatory abnormalities [36–38]. In addition, the vulnerability and sensitivity of the spinal cord after ACA/CPR are completely different from those of the brain. In partic-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, IL-4 immunoreactivity in the anterior horn was maintained after ACA/CPR (Figure 6A(f–h),B).

#### ular, the time course of cell death in the ischemic spinal cord is different from that in the 3.5.2. IL-13 Immunoreactivity

faster than brain damage.

ischemic brain after ACA/CPR [39]. This difference may be because much energy is required for the extensive activity of the motor neurons located in the anterior horn of the spinal cord [36]. It has been reported that thoracic aortic occlusion-induced spinal cord ischemia leads to neuronal damage in the ventral horn of the lumbar spinal cord from one day after spinal cord ischemia in rats [40]. In a rat model of ACA/CPR, neuronal death 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 Sham+vehicle group (Figure 6B(b–d),D).

(loss) in the anterior horn in the lumbar part of the spinal cord occurs at one day after ACA/CPR [9]. In addition, Ahn et al. [10] recently reported that neuronal death in the central nervous system (CNS) autonomic control center (myelencephalon and thoracolumbar division of the spinal cord) occurred very early compared to the other CNS divi-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 6C(f–h),D).

sions after ACA/CPR in rats. In our current study, we found that ventral motor neurons at the level of the lumbar spinal cord were dead at two days after ACA/CPR in rats. These

of the brain and spinal cord and that the spinal cord has a higher vulnerability to transient ischemia than the brain. In short, spinal cord damage following ACA/CPR occurred much

Hind-limb paralysis is one of the main disorders after ACA/CPR [8,9]. Duggal and Lach [39] reported that selective vulnerability of the lumbosacral part of the spinal cord was shown in patients with ACA/CPR and hypotension. Experimental studies on ischemic spinal cord injury have been conducted using animal models with aortic disease or local vascular change [40–42]. In a rabbit model of spinal cord ischemia, which is simply produced by occlusion of the spinal arteries that have no collateral circulation, paraplegia occurs when motor neurons in the lumbar spinal cord are damaged or dead after ischemic-reperfusion injury [43], and the death of motor neurons in the lumbar spinal cord is shown within one day after ischemia-reperfusion [44–46]. In our current study using a

## **4. Discussion**

Generally, the spinal cord is prominently susceptible to ischemic insults owing to diverse circulatory abnormalities [36–38]. In addition, the vulnerability and sensitivity of the spinal cord after ACA/CPR are completely different from those of the brain. In particular, the time course of cell death in the ischemic spinal cord is different from that in the ischemic brain after ACA/CPR [39]. This difference may be because much energy is required for the extensive activity of the motor neurons located in the anterior horn of the spinal cord [36]. It has been reported that thoracic aortic occlusion-induced spinal cord ischemia leads to neuronal damage in the ventral horn of the lumbar spinal cord from one day after spinal cord ischemia in rats [40]. In a rat model of ACA/CPR, neuronal death (loss) in the anterior horn in the lumbar part of the spinal cord occurs at one day after ACA/CPR [9]. In addition, Ahn et al. [10] recently reported that neuronal death in the central nervous system (CNS) autonomic control center (myelencephalon and thoracolumbar division of the spinal cord) occurred very early compared to the other CNS divisions after ACA/CPR in rats. In our current study, we found that ventral motor neurons at the level of the lumbar spinal cord were dead at two days after ACA/CPR in rats. These results indicated that the time course of neuronal damage/death in the CNS following global ischemia in the whole body (i.e., ACA) must be different according to the regions of the brain and spinal cord and that the spinal cord has a higher vulnerability to transient ischemia than the brain. In short, spinal cord damage following ACA/CPR occurred much faster than brain damage.

Hind-limb paralysis is one of the main disorders after ACA/CPR [8,9]. Duggal and Lach [39] reported that selective vulnerability of the lumbosacral part of the spinal cord was shown in patients with ACA/CPR and hypotension. Experimental studies on ischemic spinal cord injury have been conducted using animal models with aortic disease or local vascular change [40–42]. In a rabbit model of spinal cord ischemia, which is simply produced by occlusion of the spinal arteries that have no collateral circulation, paraplegia occurs when motor neurons in the lumbar spinal cord are damaged or dead after ischemicreperfusion injury [43], and the death of motor neurons in the lumbar spinal cord is shown within one day after ischemia-reperfusion [44–46]. In our current study using a rat model of ACA/CPR, paralysis in the hind limbs was seen one day after ACA/CPR, and most motor neurons located in the anterior horn were not seen two days after ACA/CPR. Taken together, we suggest that paraplegia following ACA/CPR might occur with motor neuron damage or death because normal motor nerve fibers (general somatic efferent) via the spinal nerves cannot innervate muscles of the limbs [47].

For several decades, RIS, as a selective monoaminergic antagonist, has been widely used for the treatment of schizophrenia [17,18]. In addition, RIS has been reported to induce hypothermia [19,21,48]. It has been reported that hypothermia can display neuroprotection and improve damaged outcomes in experimental animal models of spinal cord and brain injury [49]. However, few data concerning the effects of hypothermia against spinal cord injury after ACA have been accumulated. In this regard, we examined the effect of RIS on motor deficits in the hind limbs and its related neuronal vulnerability in the spinal cord following ACA/CPR in rats. It was reported that RIS treatment after brain transient ischemia induced hypothermia within 30 min and lasted for four hours and that hypothermia displayed effective protection against the death of hippocampal neurons induced by transient brain ischemia by attenuating glial activation and maintaining antioxidant enzymes [21]. In our present study, the effects of RIS-induced hypothermia on spinal cord injury after ACA/CPR in rats were investigated, and, as expected, RIS-induced hypothermia significantly improved paraplegia and alleviated the damage/death (loss) of ventral motor neurons at two days after ACA/CPR. These results strongly suggest that RIS treatment after ACA improves neurological dysfunction by attenuating the damage of the ventral motor neurons in patients with spinal cord injury from ACA.

Over the past few years, a body of evidence has stressed the roles of inflammation in the pathophysiology of acute brain ischemia [50]. Cytokines include many groups of

inflammatory mediators, and they act as signaling molecules to control inflammation and to induce positive or negative effects on neuronal survival [51]. It is well known that pro-inflammatory cytokines are involved in the amplification of inflammatory reactions and contribute to the pathogenesis of neurological disorders, whereas anti-inflammatory cytokines are decisively involved in resolving inflammation through downregulating the production of pro-inflammatory cytokines [52].

Some studies showed the anti-inflammatory properties of RIS in an in vivo and in vitro model. MacDowell K.S. et al. [53] demonstrated the anti-inflammatory effect of RIS. In detail, a single administration of RIS regulated various factors that triggered advanced inflammatory responses, such as the expression of inflammatory cytokines (interleukin (IL)-1β and tumor necrosis factor (TNF)-α) following lipopolysaccharide (LPS)-induced inflammation in the frontal cortex of rat brains. Additionally, a precedent study showed that RIS suppressed the production of pro-inflammatory cytokines and decreased the level of inducible NO synthase (iNOS), which are secreted by reactive microglia using a microglial cell line [54]. In this study, the immunoreactivity of pro-inflammatory cytokines (TNF-α and IL-1β) in the ventral horn of the ACA/CPR+vehicle group was increased with time after ACA/CPR, but, in the ACA/CPR+RIS group, the immunoreactivity of TNF-α and IL-1β was significantly lower than that in the ACA/CPR+vehicle group. It has been found that TNF-α and IL-1β are activated in the brains of animal models of transient brain ischemia as mediators in response to ischemic injury [55–57]. TNF-α and IL-1β play critical roles in post-ischemic inflammatory injury in the spinal cord [58–60]. Hasturk et al. [58] concluded that serum TNF-α and IL-1β levels significantly increased after spinal cord ischemia-reperfusion injury accompanied by tissue damage. In rat models of spinal cord ischemia-reperfusion injury, increased levels of cytokines induced by ischemic injury were observed to be associated with the deterioration of motor function and histological damage in the spinal cord [61] and TNF-*α* levels were significantly increased within 1.5 h, and peaked 3 h after ischemic injury [62]. In a swine model of spinal cord ischemiareperfusion injury, TNF-*α* levels were significantly increased from 6 to 24 h after ischemic injury [63]. Additionally, IL-1 expression was significantly increased in the spinal cord 6 and 36 h following ischemic-reperfusion injury in mice [64]. Taken together, we suggest that pro-inflammatory cytokines might contribute to cell death in the spinal cord following ischemia-reperfusion injury. In addition, our current findings indicate that RIS treatment after ACA/CPT induces hypothermia and prevents the abnormal expressions of TNF-α and IL-1β in the ischemic spinal cord.

In our current study, significant decreases in the immunoreactivity of anti-inflammatory cytokines (IL-4 and IL-13) were observed in the anterior horn cord in the lumbar spinal cord after ACA/CPR. However, IL-4 and IL-13 immunoreactivity in the ACA/CPR+RIS group was not reduced compared to that in the sham+vehicle group. It was demonstrated that the sustained or increased expression of endogenous anti-inflammatory cytokines (IL-4 and IL-13) contributed to neuronal survival from ischemia-reperfusion injury in the gerbil hippocampus after transient forebrain ischemia [65,66]. Additionally, some studies showed that IL-4 and IL-13 suppressed the expression and production of pro-inflammatory cytokines (TNF-α and IL-1β) in the spinal cord of animal models of spinal cord ischemia [67,68]. Therefore, taken together, the findings suggested that the maintained expression of anti-inflammatory cytokines in the ACA/CPR+RIS group may contribute to the protection of motor neurons from ACA injury.

#### **5. Conclusions**

In brief, our present findings showed that RIS treatment after ACA/CPR induced hypothermic conditions, significantly reduced mortality, and attenuated hind-limb paralysis. In addition, neuronal damage/death (loss) in the ventral horn of the lumbar spinal cord was ameliorated. These might be associated with the significant decreases of proinflammatory cytokines and the maintenance of anti-inflammatory cytokines, which might be induced by the hypothermic condition induced by RIS treatment. Taken together, we

suggest that immediate post-treatment with RIS after ACA can be utilized as a novel therapeutic approach of patients with ACA.

**Author Contributions:** Conceptualization, M.-H.W. and J.H.C.; Methodology, J.-C.L. and M.C.S.; Software, H.-I.K.; Validation, S.H. and J.H.A.; Investigation, T.-K.L., D.W.K. and H.-J.T.; Data Curation, T.-K.L. and J.H.P.; Writing—original draft preparation, T.-K.L. and J.-C.L.; Writing—review and editing M.-H.W.; Supervision, S.Y.C. and J.-C.L.; Project Administration, M.-H.W. and J.H.C.; Funding Acquisition, M.C.S., J.H.C. and S.Y.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2020R1I1A3068251, NRF-2016R1D1A1B01011790 and NRF-2019R1A6A1A11036849).

**Institutional Review Board Statement:** The experimental protocol for this study was approved (approval no., KW-200113-1; approval date, 18 February 2020) by the Institutional Animal Care and Use Committee (IACUC). The content of the protocol adhered to the guidelines that 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).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors would like to thank Seung Uk Lee and Hyun Sook Kim for their technical help in this study.

**Conflicts of Interest:** The authors declared that there are no conflict of interest to this work.

#### **Abbreviations**


## **References**

