Curcumin attenuates sensorimotor deficits induced by selective neuronal loss: A study in middle cerebral artery occlusion rat model of transient ischemic stroke.

Background: Ischemic stroke is the most prevalent and major pathological stroke type. Increased systemic oxidative stress is a crucial underlying pathophysiological mechanism of ischemic stroke. In this regard, therapeutic interventions targeting oxidative stress management would be a promising avenue. Curcumin is well recognized to counteract various pathologies effectively. The present study was designed to investigate the neuroprotective effects of curcumin against ischemia-reperfusion injury-induced selective neuronal damage. Methodology: Present study employed an ischemic brain injury model induced by middle cerebral artery occlusion (MCAO) in Wistar albino rats. Experimental groups (n=36; age-matched, female, weighing 200-240 g) were control, sham-operated, MCAO (15 min occlusion + 24 hrs. reperfusion) and curcumin-treated pre-stroke group (received curcumin 300 mg/kg body weight/day, I/P, for 30 days followed by MCAO). With the completion of the experimental protocol, sensorimotor deficits were examined by behavioral tests, including neurological deficit score, foot fault test, forelimb placing test, corner turn test, and wire hanging test. Results: Compared to MCAO rats, curcumin-treated MCAO rats showed better neurological functioning (p<0.05) as exhibited by substantial reductions in neurological score. Compared with stroke group, curcumin treated group showed better functional outcomes as displayed by improved scores in foot fault index, and forelimb placing test (p<0.01), fewer left turns (p<0.01) in corner turn test, and prolonged grip latency in wire hanging test (p<0.01). Conclusion: The present study showed that designed curcumin treatment effectively manages MCAO-induced sensorimotor dysfunction by preventing selective neuronal loss after the ischemic attack, attributed to its antioxidant, anti-apoptotic, and anti-inflammatory potentials.


Introduction
Stroke, a neurological disorder, results from compromised cerebral perfusion either because of hemorrhage or ischemia 1,2 . Approximately 85 % of all stroke cases are of ischemic origin 2 . Globally, stroke is the second leading cause of death and the third leading cause of complex disabilities with cognitive impairments 3,4 . Among ischemic stroke incidences, transient ischemic attack (TIA) accounts for 20 % of cases associated with high odds of behavioral and cognitive impairments and sensorimotor deficits 5 . Clinically, TIA is characterized by a transient episode of focal neurological signs which lasts less than 24 hrs and without tissue injury or pannecrosis (acute infarction) 6,7 . TIAs essentially result in a selective neuronal loss (SNL), a patchy (single) neuronal loss with the preserved extracellular matrix and without vascular cell death 6,8 usually injuring the hippocampus's neurons its associated areas, higher cortical regions, and striatum / sensorimotor cortex 9 . The underlying pathophysiology of ischemic stroke (IS) is based on reoxygenation (ischemia-reperfusion) injury 9 . Tissue ischemia initiates the complex sequelae of the cellular process including microglial activation, glutamate excitotoxicity, apoptosis, oxidative stress, and disintegrated blood-brain barrier. Subsequent reperfusion of ischemic tissue paradoxically initiates reactive oxygen species (ROS) production and neuroinflammation 10-12 . Hence stroke survivors specifically post-TIA, patients suffered a wide array of neurological deficits (headache, vertigo, nausea, blurred vision, convulsions, and altered consciousness), sensorimotor deficits (tremors, poor muscle coordination, and paresis or complete paralysis), and dysfunctioning of higher cortical regions (memory, speech, and other cognitive disturbances) 5 .
Therefore, post-stroke functional assessment in experimental animal models could provide a valuable understanding of neurological dysfunction's underlying pathophysiology and investigate the potential effects of therapeutic agents and rehabilitation efforts. In this regard, the middle cerebral artery occlusion (MCAO) ischemic stroke rodent model is a popular reproducible model used to study the IS-associated neuronal damage, sensorimotor deficits as well as for preclinical stroke trials 13 .
Two treatment approaches are currently available for ischemic stroke patients, intravenous thrombolysis and endovascular (mechanical) recanalization 1,3 . For the former treatment option, the only approved drug is tissue plasminogen activator (tPA), which has a minimal therapeutic window of 3 to 4.5 hrs 14 , and for the latter, very few patients qualify for mechanical thrombectomy 1,3,15 due to these therapeutic tribulations, pharmacological interventions are limited so far.
Among nonpharmaceutical interventions, natural products have long been proven as potential therapeutic agents attributable to their antioxidant properties 14,16 . Turmeric or Curcuma longa Linn is a well-known culinary spice and a medicinal herb used in the traditional medicine system for decades. Curcumin, a bioactive constituent, extracted usually from the turmeric plant roots reported to possess antioxidants, anti-apoptotic, anti-inflammatory, anti-diabetic, and hypolipidemic therapeutic potentials 17-18 hence known to exert protective effects on various physiological systems including the nervous system 16,19,20 . Therefore, this study aimed to investigate the neuroprotective effect of curcumin administration against SNL-induced sensorimotor deficits in the MCAO rat model of transient focal cerebral ischemia.

Study Design
Experimental animal research study conducted at the Department of Physiology, University of Karachi.

Ethics Statement
The departmental research committee approved all experimental procedures, Department of Physiology, University of Karachi, in accordance to the National Institutes of Health's Guide for the care and use of laboratory animals, and are reported according to the ARRIVE (Animal Research: Reporting of in Vivo Experiments) guidelines 21 .

Experimental Animals
Female Wistar albino rats (body weight: 220 ± 20 g, age: 5-6 weeks old), purchased from International Centre of Chemical and Biological Sciences, University of Karachi (Karachi, Pakistan). Animals were housed at Animal Care Facility, Department of Physiology, University of Karachi. Animals kept under 12 hrs light / 12 hrs dark cycle, temperature-controlled (23 ± 2°C), ventilated room and fed with standard laboratory diet and water ad libitum.

Experimental Groups
A total of 36 animals were used in this study. After one week of acclimatization, animals were randomly divided into four experimental groups as:  Control group (n=9)  Sham-operated group (n=9)  MCAO group (n=9), stroke-induced group  MCAO+CUR group (n=9), stroke-induced group pre-treated with curcumin All animals received a standard laboratory diet and water ad libitum throughout the study protocol. Animals from all experimental groups were evaluated for sensorimotor changes at the end of the experimental period (24 hrs. after MCAO and sham surgery).

Treatment Protocol
Curcumin dose was prepared according to the previously described method 22 . The MCAO+CUR group's animals receive curcumin 300 mg/kg body weight, dissolved in normal saline (pH 7.4), and administered intraperitoneally for 30 days. On the 31st day, animals were subjected to MCAO for 15 min, followed by reperfusion of 24 hrs.

MCAO Protocol
Transient MCAO was induced via the monofilament method as described in detail by previous studies 23,24 .

Anesthesia
Animals were anesthetized intraperitoneally with a mixture of xylazine (5 mg/kg) and ketamine hydrochloride (50 mg/kg) 23 . Anesthetic depth was confirmed with the absence of a paw-pinch reflex. Body temperature was maintained at 37.0 ± 0.5°C, and other body conditions were monitored perioperatively.

MCAO surgery
For MCAO induction, left common, external, and internal carotid arteries were exposed after ventral midline cervical incision. Then a 4-0 monofilament silicon-coated suture with a rounded tip of 0.37 mm diameter was inserted at the bifurcation of the left common carotid artery and advanced until the monofilament occluded the left middle cerebral artery (MCA). After 15 minutes of occlusion, the monofilament was withdrawn to allow for reperfusion (24 hrs.). The neck skin incision was sutured back, and the animal was allowed to recover 10,25 .

Sham surgery
Sham-operated animals receive the same MCAO surgery, including the exposure of all carotid arteries without monofilament insertion and occlusion of the middle cerebral artery. *Of note: control, sham and MCAO groups were based on previous literature 25 .

Neurobehavioral Testings
At the end of the study protocol, all experimental groups were examined for MCAO-induced SNL associated sensorimotor changes using a battery of different neurobehavioral tests.
Neurobehavioral tests used in this study include neurological deficit score, foot-fault test, vibrissae evoked forelimb placing test, corner turn test, and wire hanging test.

Procedures for Neurobehavioral Tests
All neurobehavioral testings were done by qualified persons blinded to experimental groups. To ensure accurate neurobehavioral results, animals were handled and trained for one week before the baseline testing. Also, all the training and testings were done in the animals' housing cage and in the light phase (approx. same time) daily.

Neurological Deficit Score
Neurological deficit (ND) score is a composite scoring used to reveal the ischemic stroke associated neurological deficits in animals 26 . NDscore has been performed 24 hrs. post-MCAO. This neurological battery consists of the following three tests: (1) Body Twisting: test postural reflex (2) Forelimb Flexion: test sensorimotor integration (3) Balance Test: test limb dysfunction The overall composite score is based on predetermined criteria 26 . Total ND-score graded on a scale of 0 -9. Where 0 represents normal, and 9 represents the maximum neurological deficit as described 26 .

Foot Fault Test
The foot fault asymmetry test is commonly used to evaluate the post-stroke motor coordination and sensorimotor functioning and rehabilitation effects in MCAO experimental models 13 . The foot fault test is a skilled task in which animals were trained to walk on an elevated grid to record the number of foot faults (limb slip) as rats with ischemic brain damage exhibit greater paw-misplacements than normal rats 27 so the total number of foot-faults for forelimb and hindlimb were recorded as and scored as foot fault index (FFI).
FFI= (contralateral faults -ipsilateral faults) ÷ (total steps). The FFI rated on the scale as zero indicates no asymmetry, the score of the negative number indicates ipsilateral deficit while the positive score represents contralateral deficit 27 .

Vibrissae Evoked Forelimb Placing Test
This test allows us to study the focal cerebral ischemia associated with neurological dysfunction and the effect of treatment interventions on poststroke rehabilitation 28 . For this task, the animal was lifted by torso to freely hang in the air as its forelimbs and hindlimbs freely hung in the air. For the induction of ipsilateral and contralateral forelimb responses, the animal's vibrissae were brushed at the edge of a table. In response, animals (without any brain injury) place a forelimb ipsilateral to vibrissae-evoked side while MCAO animals with the motor deficit would exhibit impaired limb placement 28 . Therefore, animals were tested for two sub-types of vibrissae-evoked forelimb placing test.

Corner Turn Test
Corner turn (CT) test, a well-known sensorimotor test to identify the functional deficits and sensorymotor (postural) asymmetries after ischemic stroke. This test examined the rodent's reliance on a preferential limb and so turned in a specific direction 29 . In this test, the animal was placed and allowed to reach the corner and noted the turns (left or right) taken by the animal either by place one or both forelimbs. Animals with MCAO damage exhibit reliance on a non-impaired limb and turning in an ipsilateral direction. The animal underwent 10 trials, and a reliant turning side was noted. Results scored as % = [(Number of ipsilateral (left) turns) ÷ (Total number of right turns + left turns) × 100] 29 .

Wire Hanging Test
This is a simple test used to evaluate the post-MCAO motor functioning based on animal strength of grip, endurance, balance, and sensorimotor coordination. Animals with ischemic brain injury show poor performance in wire holding task and have very short latency of fall 26,30 . In this task, the animal was allowed to hang on a wire, and the wire holding time (latency to fall) was noted 30,31 . The increased wire holding time indicates better grip strength and muscle endurance of animals.

Statistical Analysis
Data were tabulated and expressed as mean ± standard error of the mean (SEM); values in percentage (%) are presented as mean ± standard deviation (SD). Statistical analysis was performed using statistical software SPSS 17.0. Intergroup differences were tested with ANOVA followed by post hoc t-test. P < 0.05 was considered statistically significant.

Results
Animals from all groups showed normal behavioral results before MCAO or sham surgery. Table 1 showed the comparison of neurobehavioral tests among different experimental groups.

Neurological Deficit Score
The MCAO group showed a significantly higher neurological deficit score (7.83 ± 0.47) as exhibited by impaired neurological performance compared to control and sham groups.

Foot-Fault Test
As shown in Table 2, animals from the control and sham groups showed no foot faults. MCAO group exhibit poor performance in foot fault task. MCAO group rats scored higher for hind limb than forelimb as exhibited by higher scores (FFIHindlimb = 0.15 ± 0.042; FFIForelimb = 0.1 ± 0.025), indicating that MCAO affects contralateral hindlimb more than contralateral forelimb.

Vibrissae evoked Forelimb Placing Test
Among all other experimental groups, rats from the MCAO group showed a significantly higher deficit for the forelimb placement test. Also, the MCAO group scored significantly (p < 0.001) higher for contralateral forelimb than ipsilateral forelimb compared to the control group (Table 3).

Corner Turn Test
As displayed in Table 4, animals from the control group did not show corner turn asymmetries as exhibited by approx. equal number of left and right turns (average left turns 53.3 ± 0.852 % and right turns 46.7 ± 0.816 %). Similarly, sham-operated rats also did not exhibit any significant limb reliance. The MCAO group showed significantly higher percentage of ipsilateral (left) turns (71.66 ± 0.752 %) as compared to control (p < 0.01) and sham-operated (p < 0.01) groups exhibiting corner turn asymmetry and reliance on non-impaired (left) limb. In MCAO+CUR treated group; decreased % ipsilateral turns (66.66 ± 1.211 %) were noted as compared to MCAO group (p > 0.05).

Wire Hanging Test
Compared to the control and sham-operated groups, animals of the MCAO group exhibit poor wire holding as displayed by significantly decreased wire-holding time (15.5 seconds) (p < 0.001 for control vs. MCAO) and (p < 0.001 for sham vs. MCAO) groups (Table 5). Curcumin pre-treated animals (MCAO+CUR) showed significantly better wire holding time (23.5 seconds) when compared to the MCAO untreated group (p < 0.01).

Discussion
Globally, stroke is the single major cause of disabilities affecting around 15 million people 7 . Ischemia-reperfusion mediated oxidative stress initiates the myriad of pathological processes that ultimately lead to neuronal death and tissue necrosis 9 . Due to these catastrophic cellular events, patients suffer devastating sensorimotor and cognitive impairments after ischemic stroke 32 . Furthermore, very limited options are available to treat ischemic stroke survivors. Hence identification of an effective management approach, including treatment with preventive measures is a challenging and urgent need 3 . Herbs and natural products have been long recognized for neuroprotective potentials mainly attributable to their antioxidant and anti-inflammatory effects 5 . Therefore, the present study aimed for functional assessments in the MCAO rats under curcumin pretreatment. MCAO-induced regional cerebral injuries exhibited by sensorimotor dysfunction. This study includes various behavioral testings to evaluate the transient ischemia associated with neurological dysfunction and the treatment regime's effect (300 mg curcumin/kg bodyweight for 30 days) in MCAO induced rats.
Results of the present study also displayed the functional deficits in the MCAO group as exhibited by significantly higher ND score (p < 0.005), high foot fault index, poor grip and memory, impaired forelimb placing (p < 0.005), wire-hanging (p < 0.005) and corner turn (p < 0.01) tests when compared with normal animals. The previous studies 6,33 also reported the MCAO associated sensory, motor, and cognitive deficits principally because of ROS-mediated apoptosis and necrosis cascade and neuroinflammation. PPARγ is an anti-inflammatory transcription factor hence mediate neuroprotection against ischemiareperfusion injury 37 .
Another experimental study also reported that curcuminoid pre-treatment significantly reduces cerebral edema and the size of ischemic infarct in the rat model of transient-MCAO 38 . Curcumin supplementation significantly reverses the poststroke oxidative stress and neurological dysfunctions as exhibited by improved neurobehavioral tests, attributed to curcumin antioxidant potentials 38 . Antioxidant potentials of curcumin include a potent scavenger of free radicals, increases endogenous antioxidants enzymes, and modulates γ-enolase pathways 19,35 .

Conclusion
Taken together, the data of the present study indicate that transient ischemic stroke results in a selective neuronal loss that impaired neurological functions. Designed curcumin treatment regime found to be effective against the sensory, motor, and cognitive functions in the MCAO rat model of ischemic stroke. Curcumin mediated neuron survival principally by managing oxidative tissue damage.