Pharmacological connection of Histamine-1 (H1) Receptor Mediated Neuroprotective mechanism of Ischemic preconditioning in rat

Prabhat Singh¹, Bhupesh Sharma^2,3*

¹Ph.D Student, Neuropharmacology Lab., Department of Pharmacology, KSCP,

Subharti University, Meerut, Uttar Pradesh, India.

²Professor, Department of Pharmacology, Amity Institute of Pharmacy,

Amity University, Sector-125, Noida - 201313, Uttar Pradesh, India.

³Chief Consultant, CNS Pharmacology, Conscience Research, Pocket F- 233,

B (Near Sai Vatika), Dilshad Garden, Delhi - 110095 India.

*Corresponding Author E-mail: sharmaslab2@gmail.com, bsharma5@amity.edu, drbhupeshresearch@gmail.com

ABSTRACT:

Cerebral ischemia and ischemia-reperfusion is an essential contributor to acute cerebral stroke. Ischemic preconditioning (IPC) has been proven to provide neuroprotection in ischemia-reperfusion injury in rats, but their mechanism behind neuroprotection in cerebral stroke are still unclear. Central histaminergic pathway has crucial role in the pathogenesis of cerebral stroke, but their neuroprotective role in IPC is still unidentified. This research explores the role of histamine-1 receptor in IPC induced neuroprotection against ischemia-reperfusion induced cerebral injury. Rat were subjected to 17 min of global cerebral ischemia (GCI) by occluding both carotid arteries followed by reperfusion for 24 h, to produce ischemia-reperfusion induced cerebral injury. TTC staining was used to measure cerebral infarct size. Morris water maze test was used to assess memory. Inclined beam-walk, hanging wire, lateral push and rota-rod tests were used to assess degree of motor incoordination. Brain acetylcholinesterase activity, nitrite/nitrate, glutathione, TBARS and MPO levels were also examined. GCI has produced a significant increase in cerebral infarction, brain nitrite/nitrate, MPO, TBARS and AChE activity along with a reduction in glutathione content. Impairment of memory and motor coordination were also noted in GCI induced rat. IPC was employed that consist of 3 preceding episodes of ischemia (1 min) and reperfusion (1 min) both immediately before GCI significantly decreased cerebral infarction, motor incoordination, memory impairment and biochemical impairment. Pretreatment with L-histidine mimicked the neuroprotective effects of IPC. L-histidine induced neuroprotection were significantly abolished by chlorpheniramine, a H1 receptor antagonist. We conclude that neuroprotective effects of IPC, probably occurs through the central histaminergic pathway, and histamine-1 receptor could be a new target behind the neuroprotective mechanism of IPC.

KEYWORDS: Histamine, Chlorpheniramine, Brain stroke, Cerebral ischemia, Brain infarction, Cognition, Motor impairment.

INTRODUCTION:

Cerebral stroke is the most common form of cerebrovascular disease and it is the leading cause of public health problem, with high prevalence of death and disability. Cerebral stroke is a condition characterized by rapid onset of neurological damage due to disruption of cerebral blood^1,2.

An attempt to attenuate this injury led to the discovery of phenomenon like ischemic preconditioning (IPC) in heart and brain^3,4. IPC is an adaptive endogenous neuroprotective concept in which prevents the subsequent deleterious effects of ischemia-reperfusion injury on that organ^4,5. The protective strategies of IPC have been studied to resist injuries against hypoxia and in neurodegenerative disorders like Alzheimer’s disease⁶. Mechanism involved in protective effects of preconditioning are complex and interlinked, which could either promotes antioxidant system, anti-inflammatory cascades or inhibits the synthesis of proinflammatory cytokine and increase the resistance of cells or tissues against a subsequent, more rigorous ischemic event⁷. Preconditioning has induced angiogenesis, maintain vascular endothelial function and increases cerebral blood flow, that help to reduce cerebral damage ischemic stroke⁸. In last few years, clinical trials have shown that IPC can effectively induces cerebral ischemic tolerance, thus reducing ischemia-reperfusion injury. However, the underlying mechanism is not fully understood.

Histamine is a neurotransmitter in the central nervous system (CNS), acting through histaminergic receptors like H1 and H2 receptors in the brain⁹. H1 receptors are mainly expressed on neurons and astrocytes that control various physiological functions such as anxiety, learning, memory, and stress¹⁰. Alterations of histamine levels in brain are related to CNS dysfunction and neurodegenerative disorders¹⁰. Histamine plays an important role in the pathogenic progression after cerebral ischemia. Further reports suggest that histamine suppresses the release of glutamate, inhibits excitotoxicity and inflammation. It also helps to restore cerebral blood flow and promote neurogenesis¹¹. Previous studies have provided crucial insights into the role of H1 receptor signaling in CNS functions, but it remains unclear that how H1 receptor dependent signaling affects ischemic tolerance in cerebral ischemic preconditioning. To perceive their role in this exploration we have employed Histaminr-1 receptor modulator (L-histidine and chlorpheniramine) during IPC and ischemia-reperfusion induced brain injury.

MATERIALS AND METHODS:

Animals:

Wistar albino rats (male; 200 to 250g) were purchased from the Indian Veterinary Research Institute, India and maintained on standard laboratory conditions with free access to water. The protocol was certified by Institutional Animal Ethics Committee. The care of experimental animals was done as per the recommendations of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India (Reg. No. 1147/ab/07/CPCSEA).

Induction of global cerebral ischemia (GCI):

Bilateral carotid artery occlusion (BCAO) was used to establish GCI in rats, according to methods described by Rehni and Singh, 2012. Animals were sedated with chloral hydrate (400mg kg^-1; i.p.) and maintained their body temp close to 37°C. A middle incision was applied in the neck, and then both carotid arteries were separated from nearby tissues. A cotton strand was passed below the carotid arteries and GCI was provoked by obstruction of these arteries for 17 min and then reperfusion was allowed for 24 h, and the cut was stitched back. The experimental rats were shifted individually to their plastic cage and were let to recover².

Induction of cerebral ischemic preconditioning (IPC):

Cerebral ischemic preconditioning in animals was induced by application of three ischemic episodes of 1 min then reperfusion of 1 min, just earlier bilateral carotid artery occlusion for 17 min².

Drugs and experimental protocol:

All drug and chemicals were purchased from standard vendors. The dosing route and scheme were chosen according to formerly recognized investigations from various testing centers. Total six groups were used in this study and each group consisted of eight Wistar albino rat.

Group I: Sham control: Each rat was exposed to the operative process and cotton strand was passed below both the carotid arteries but occlusion was not performed. After 17 min thread was taken out and the animal was stitched and allowed to recover health for 24 hours.

Group II: Ischemia-reperfusion (control): Each rat was subjected to 17 min of global cerebral ischemia (GCI) and then reperfusion for 24 hours.

Group III: IPC: Rats were exposed to three episodes of ischemia (1 min) then reperfusion of every 1 min. This was immediately pursued before GCI (17 min) as well as reperfusion of 24 h.

Group IV and V: Chlorpheniramine (dose 1 and dose 2) and IPC: Chlorpheniramine (15 and 30mg kg^-1; i.p.) was administered 1 hr before carotid arteries occlusion. Remaining procedure was similar to the group III.

Group VI: L-histidine and IPC: L-histidine (1,000mg kg^-1; i.p) was administered 1 hr before carotid arteries occlusion. Remaining procedure was similar to the group III.

Behavioral assessments:

Hanging wire test:

This test is employed to check grasping power and strength of forelimbs of rats¹². The rats were hung drooped on a cord expanded between two poles (at height of 45cm from the foam slip) by forelimbs. The fall-off time (sec) was noted down. Cut-off time was set for 2 min.

Rota rod:

Grip strength and motor coordination were assessed using rota-rod apparatus as per the previously published procedures. Falling time latency of animals were noted by a skilled observer unsighted to the experimental procedure^1,2.

Inclined beam walking:

The test was used to assess the coordination of hind and forelimbs. The motor performance of rat was scored on a scale (zero to four). This test was conducted before GCI and after 24 h ischemia-reperfusion as per the previously published procedures².

Lateral push

Animal was kept on an irregular surface for stronghold and was evaluated for resistance to lateral push from either side of the shoulder. This test was carried out prior to GCI and after 24 h ischemia-reperfusion as per the previously published procedures².

Assessment of memory using morris water maze (MWM):

Learning and memory was evaluated using MWM, as per, previously published procedures². Escape latency time-ELT on 4th test day was recorded as an indicator of learning. Afterward animals were exposed to GCI and reperfusion. Times spent in the target quadrant (TSTQ) was recorded, is an indicator of memory on 5^th test day ²(Rehni and Singh, 2012).

Assessment of cerebral infarct Size:

Previous studies have demonstrated that GCI and reperfusion induces brain damage², which was assessed by TTC (2-3-5-triphenylterazolium chloride) staining, according to the previously published procedure¹³.

Dissection and homogenization:

Dissection, homogenization and isolation of the brain were done according to the previously published procedures¹³. The supernatants were separated for biochemical estimations.

Biochemical estimations:

Estimation of brain lipid peroxidation, brain nitrite/nitrate, reduced glutathione (GSH), myeloperoxidase (MPO), acetylcholinesterase (AChE activity) and total protein in brain:

Thiobarbituric acid reactive substances (TBARS) at 532 nm, nitrite/nitrate at 545nm, reduced GSH content at 412 nm, Brain MPO at 460nm, AChE activity at 420 nm, total protein at 750nm, were estimated spectrophotometerically (UV-1800 ENG 240V; Shimadzu Corporation Japan), as per the previously published procedure^1,13.

Statistical analysis:

The results were expressed as mean ± standard error of means (S.E.M.). Statistical analysis for all the results were done using one-way ANOVA followed by Tukey's multiple range tests. A value of p ≤0.05 was considered to be statistically significant.

RESULTS:

Table 1: Effect of ischemic preconditioning on motor performance of the animals

Group	Rota Rod (Fall down time in sec)		Inclined beam walks (Scores of motor incoordination)		Lateral push (Percent resistance to lateral push)		Hanging wire test (Latency to fall or Mean fall time in sec)
Group	Basal	Final	Basal	Final	Basal	Final	Basal	Final
Sham	280 ± 13.44	276 ± 17.94	0.45 ± 0.01	0.47 ± 0.024	100 ± 4.1	100 ± 4.7	87 ± 4.45	84 ± 5.63
IR (control)	280 ± 13.44	83 ± 5.39^a	0.44 ± 0.01	3.7 ± 0.18^a	98 ± 4.01	23 ± 1.08^a	86 ± 4.40	45 ± 3.01^a
IPC	274 ± 13.15	203 ± 13.19^b	0.45 ± 0.01	2.3 ± 0.117^b	99 ± 4.06	71 ± 3.34^b	90 ± 4.60	60 ± 4.02^b
CD1 +IPC	278 ± 13.34	90 ± 5.85^c	0.49 ± 0.015	2.8 ± 0.143^c	97 ± 3.97	38 ± 1.78^c	86 ± 4.40	55 ± 3.68^c
CD2 +IPC	277 ± 13.29	86 ± 5.59^c	0.47 ± 0.01	3.4 ± 0.174^c	99 ± 4.05	31 ± 1.45^c	89 ± 4.56	49 ± 3.28^c
H + IPC	273 ± 13.10	241 ± 15.66^c	0.42 ± 0.013	1.7 ± 0.087^c	93 ± 3.81	80 ± 3.76^c	84 ± 4.30	68 ± 4.55^c

Results of each group were represented as mean ± S.E.M. IR: Ischemia-reperfusion; H: L-histidine; C: Chlorpheniramine; IPC: Ischemic Preconditioning; D1: Dose 1; D2: Dose 2. Rota Rod: F(5, 47)= 78.023; ^a p<0.05 vs sham, ^b p<0.05 vs control, ^cp<0.05 vs IPC. Inclined beam walk: F(5, 47)= 75.071; ^a p<0.05 vs sham, ^b p<0.05 vs control, ^cp<0.05 vs IPC. Lateral push: F(5, 47)= 108.457; ^a p<0.05 vs sham, ^b p<0.05 vs control, ^cp<0.05 vs IPC. Hanging wire test: F(5, 47)= 18.118; ^a p<0.05 vs sham, ^b p<0.05 vs control, ^cp<0.05 vs IPC.

Table 3: Effect of ischemic preconditioning on various biochemical changes

Group	Brain nitrite/nitrate (μg/mg protein)	Brain GSH (nM/mg of protien)	Brain TBARS nM/mg protein)	Brain MPO (mU/g of protein)	Brain AChE (μM of ACh hydrolyzed/min/mg of protein)
Sham	12.5 ± 0.58	32.1 ± 1.04	23.3 ± 1.25	29.1 ± 1.54	42 ± 0.89
IR (control)	29.57 ± 1.38^a	17.3 ± 0.56^a	72.3 ± 3.90^a	80.3 ± 4.25^a	165 ± 3.49^a
IPC	18.6 ± 0.87^b	26.3 ± 0.85^b	36.4 ± 1.96^b	42.2 ± 2.24^b	77.3 ± 1.64^b
CD1 +IPC	23.3 ± 1.09^c	20.4 ± 0.66^c	68.9 ± 3.72^c	74.3 ± 3.94^c	160.7 ± 3.40^c
CD2 +IPC	27.5 ± 1.29^c	18.7 ± 0.61^c	70.4 ± 3.80^c	77.8 ± 4.12^c	162.7 ± 3.45^c
H + IPC	15.8 ± 0.74^c	30.1 ± 0.98^c	28.4 ± 1.53^c	37.3 ± 1.98^c	62.3 ± 1.32^c

Results of each group were represented as mean ± S.E.M. H: L-histidine; C: Chlorpheniramine; IPC: Ischemic Preconditioning; IR: Ischemia-reperfusion;

GSH-glutathione; MPO-myeloperoxidase; AChE- acetylcholinesterase; ACh- acetylcholine; TBARS-thiobarbituric acid reactive substances

Brain nitrite/nitrate: F(5,47)= 23.734; ^a p<0.05 vs sham; ^b p<0.05 vs control; ^c p<0.05 vs IPC.

Brain GSH: F(5,47)= 18.229; ^a p<0.05 vs sham; ^b p<0.05 vs control; ^c p<0.05 vs IPC.

Brain TBARS: F(5,47)= 63.501; ^a p<0.05 vs sham; ^b p<0.05 vs control; ^c p<0.05 vs IPC.

Brain MPO: F(5,47)= 28.532; ^a p<0.05 vs sham; ^b p<0.05 vs control; ^c p<0.05 vs IPC.

Brain AChE: F(5,47)= 66.432; ^a p<0.05 vs sham; ^b p<0.05 vs control; ^c p<0.05 vs IPC.

Figure 2: Effect of ischemic preconditioning on cerebral infarction

GCI produced a significant (p < 0.05) increase in cerebral infarction in control group when compared to the sham group. IPC significantly (p < 0.05) attenuated ischemia-reperfusion induced rise in cerebral infarction. Pretreatment of chlorpheniramine significantly (p < 0.05) abolished IPC-induced reduction of cerebral infarction. Pharmacological preconditioning of L-histidine reduces cerebral infarct size when compared to the IPC group. Each group represents mean ± S.E.M. IR: Ischemia-reperfusion; H: L-histidine; C: Chlorpheniramine; IPC: Ischemic Preconditioning; IPC: Ischemic Preconditioning; ^a P<0.05 vs sham, ^b P<0.05 vs control, ^c P<0.05 vs IPC: preconditioning

DISCUSSION:

In this study, we investigate the neuroprotective role of ischemic preconditioning (IPC) against ischemia-reperfusion induced cerebral injury. We found that ischemia-reperfusion has impaired motor coordination, cognitive function and increases cerebral damage (TTC stain) in rats. The biochemical profile showed elevation in brain nitrite/nitrate, TBARS, MPO, and AChE with reduction in GSH levels in ischemia-reperfusion treated rats. These findings are consistent with previous studies showing symptoms of cerebral ischemia in rodents^1,14. These outcomes indicate that IPC produced a significant neuroprotective effect, just before bilateral carotid artery occlusion for 17 min. IPC has improved motor and cognitive dysfunction and impaired biochemical parameters in ischemia-reperfusion induced cerebral injury. IPC mediated neuroprotective effects have been significantly abolished by pretreatment with chlorpheniramine (H1 receptor antagonist). This is the first study which reports the neuroprotective role of IPC is may be due to modulation of histaminergic (H1 receptor) receptors.

Global cerebral ischemia employed in this study is well established model to simulate the clinical condition of cerebral ischemia¹. Ischemia-reperfusion has reduced cerebral blood flow in CA₁ region of the hippocampus and causes injury to CA₁ cells, which takes part in cognition as well as social behavior^1,15. Cholinergic system in brain alters during and after cerebral ischemia¹⁶, which plays an important role in learning and memory¹⁷. Cerebral ischemia has impaired brain histaminergic system that affects normal brain function. Activation of central histaminergic activity prevents the progression of ischemia-reperfusion brain injury¹⁸. Therefore, in this study, we employed the Morris water maze test to assess cognitive functions and lateral push test, inclined beam walk test, hanging wire and rota-rod test for the evaluation of motor function.

IPC provide neuroprotection against cerebral ischemia. IPC has been reported to reduce AChE activity and maintains acetylcholine level in brain, which is essential for cognitive function¹⁹. Expression of H1 receptors are known to be an important for long-term potentiation and memory in the hippocampus¹⁰. Previous reports suggest that treatment of L-histidine increases the concentration of histamine in brain and it helps to improve cognitive function by stimulating histaminergic pathway in brain²⁰. H1 receptor antagonist like chlorpheniramine has abolished the protective effects of l-histidine that were mediated via histaminergic pathway²¹. The results of present investigation indicate that IPC has improved various behavioral dysfunctions via modulating H1 receptors, while their antagonist like chlorpheniramine abolished neuroprotective effects of IPC in ischemia-reperfusion, due to blockade of H1 receptor in brain.

Increased lipid content together with the reduced antioxidant enzymes in the brain make it highly susceptible to generate oxidative free radicals, which play a crucial role in the brain damage occurred during ischemia-reperfusion. This oxidative stress induces impairment in brain GSH, lipids and proteins, leading to alteration of the brain function and cell death²². Previous findings suggest that excessive accumulation of glutamate in extracellular fluid and overstimulation of N-methyl-d-aspartate (NMDA) receptors causes neuronal cell death in the early phase of cerebral ischemia^11,23,24. Cerebral ischemia has been reported to downregulate the expression of GLT-1 (Glutamate transporter-1), is essential for maintaining the appropriate concentration of extracellular glutamate, and it helps to prevent excitotoxicity of glutamate in brain²⁴. The extents of oxidative damage were evaluated by estimating the level of TBARS, GSH, and inflammation in brain tissues as well as brain nitrite/nitrate content. Neuronal cell death was assessed by TTC satin.

IPC is a form of cellular adaptation against ischemia-reperfusion induced oxidative stress and it helps to prevents delayed neuronal cell death in the rodent hippocampus CA1 region after a severe ischemic injury. Protective mechanism of IPC is closely related to NMDA receptor against glutamate excitotoxicity²³. Preconditioning has been reported to prevent neurons against exogenous glutamate toxicity and ameliorate brain damage after cerebral ischemia, which might be due to the upregulation GLT-1^25,26. Histamine increases the expression of GLT-1 via stimulating H1 receptors in brain, thus it helps to provide neuroprotection against excitotoxicity and ischemic injury²⁷. L-Histidine, a precursor of histamine, abolished neuronal injury in cerebral ischemia and suppresses the increased glutamate content during ischemia^20,27. Previous reports suggest that blockade of histamine-1 receptor by chlorpheniramine improved the excitotoxic response of NMDA receptors, which is suppressed by histamine^11,28. Therefore, modulation of histamine-1 receptor is capable of restoring oxidative stress induced brain damage.

CONCLUSION:

The data in this research study confirmed that IPC may provide protection against ischemia-reperfusion induced cerebral injury, which might be through the modulation of H1 receptors. This study exposes that IPC is an effectual concept to ameliorate ischemia-reperfusion provoked difficulties via H1 receptors modulation. Therefore, the remedial properties of IPC may be employed in cerebral ischemia-reperfusion injury and its associated condition such as cerebral stroke.

ACKNOWLEDGMENTS:

Authors are grateful to Dr. Nirmal Singh (Faculty of Medicine from Punjabi University-Patiala) for his priceless recommendations. We are also grateful to Prof. V. K. Sharma (Bharat Institute of Technology-Meerut) along with the SPRFCT, Bharat Institute of Technology-Meerut for offering to deliver all the required services.

CONFLICT OF INTEREST:

Author has no conflicts of interest to publish this research paper.

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Received on 16.05.2020 Modified on 18.06.2020

Research J. Pharm. and Tech. 2021; 14(5):2717-2722.

DOI: 10.52711/0974-360X.2021.00479

Group	Morris water maze (MWM)
	Escape latency time (sec)
	Day 1	Day 4
Sham	108 ± 0.032	39 ± 2.184^a
IR (Control)	107 ± 3.456	37 ± 2.072^a
IPC	105 ± 3.424	40 ± 2.24^a
CD1 +IPC	103 ± 3.36	39 ± 2.184^a
CD2 +IPC	109 ± 3.296	43 ± 2.408^a
H + IPC	102 ± 3.488	40 ± 2.24^a