Neuroprotective Effects of Theobromine in permanent bilateral common carotid artery occlusion rat model of cerebral hypoperfusion

Cerebral hypoperfusion (CH) is a common underlying mechanism of dementia disorders linked to aberrations in the neurovascular unit. Hemodynamic disturbances adversely affect cellular energy homeostasis that triggers a sequence of events leading to irrevocable damage to the brain and neurobehavioral discrepancies. Theobromine is a common ingredient of many natural foods consumed by a large population worldwide. Theobromine has shown health benefits in several studies, attributed to regulation of calcium homeostasis, phosphodiesterase, neurotransmission, and neurotrophins. The current study evaluated the neuroprotective potential of theobromine against CH in the permanent bilateral common carotid artery occlusion (BCCAO) prototype. Wistar rats were distributed in Sham-operated (S), S + T100, CH, CH + T50, and CH + T100 groups. Animals received permanent BCCAO or Sham treatment on day 1. Theobromine (50, 100 mg/kg) was given orally in animals subjected to BCCAO for 14 days daily. CH caused neurological deficits (12-point scale), motor dysfunction, and memory impairment in rats. Treatment with theobromine significantly attenuated neurological deficits and improved sensorimotor functions and memory in rats with CH. In biochemistry investigation of the entire brain, findings disclosed reduction in brain oxidative stress, inflammatory intermediaries (tumor necrosis factor-α, interleukin-1β and − 6, nuclear factor-κB), markers of cell demise (lactate dehydrogenase, caspase-3), acetylcholinesterase activity, and improvement in γ-aminobutyric acid quantity in rats that were given theobromine for 14 days daily after CH. Histopathological analysis substantiated attenuation of neurodegenerative changes by theobromine. The findings of this study indicated that theobromine could improve neurological scores, sensorimotor abilities, and memory in CH prototype.


Introduction
Pre-existing cardiovascular (e.g., heart failure, cardiac arrest, high blood pressure) and metabolic disorders (e.g., atherosclerosis and diabetes) cause hemodynamic changes in the brain that forms the basis of several progressive neurocognitive disorders (Zhao and Gong 2015). Abnormality in autoregulatory mechanisms of cerebral blood flow (CBF) causes a hypoperfusion state, resulting in inefficient

Experimental subjects
The IAEC permitted the investigation procedure (Ref. no. SSP/IAEC/2019/009 on date 17-11-2019), and animal experimentations were performed as per the guiding ethics of CPCSEA, GOI (New Delhi). Wistar rats (9-10-month adults) of male sex (body weight range 230 ± 10 g) were acquired from AIIMS, New Delhi (India), acclimatized for 14 days, and later subjected to experiments between 0900 and 1600 h period. CPCSEA registered institutional establishment (Regd. no. 1616/PO/Re/S/12/CPCSEA) was utilized to house rats in separate cages (polyacrylic) under an archetypal laboratory environment. The animal caretakers were kept blinded to the various drug regimens.

Cerebral hypoperfusion
The standard technique of bilateral common carotid artery occlusion (BCCAO) was applied to initiate CH in rats (Bhuvanendran et al. 2019;Yanpallewar et al. 2005). Initially, atropine sulfate (0.5 mg/kg, i.p.) was given in rats before ketamine (90 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) injections, and subsequently, reflexes were gauged to ascertain anesthesia. A ventral midline cut was given in the neck (in the middle of the neck and sternum) between sternocleidomastoid and sternohyoid muscles parallel to the trachea. Common carotid arteries (CCA) were identified adjacent to the sternocleidomastoid muscle, and both right and left CCA was bisected carefully from the adventitial sheath and vagosympathetic nerve. A 3 − 0 silk suture (TRUSILK®) was sterilized, and both CCAs were double-ligated. Ligation of 1st carotid, either left or right, was swapped throughout this procedure (Soria et al. 2013). The skin was sutured and was applied with Neosporin®. Body temperature (37 ± 0.5 °C) was maintained throughout the surgery, including the recuperative period where free access to pulverized pellet diet and water was permitted. Rats displaying post-surgery-induced hesitancy in water intake were administered buprenorphine (0.05%, i.p) once (Saxena et al. 2015).
In contrast to caffeine, the psychostimulant effect of theobromine lacks dependence and withdrawal (Jacobson et al. 2020;Monteiro et al. 2016;Meredith et al. 2013). Theobromine has good lipophilicity, penetrates biological barriers, and has a greater plasma half-life relative to caffeine that accounts for better therapeutic outcomes in many disorders (Martinez-Pinilla et al., 2015). Theobromine has mild adverse effects and a high therapeutic index relative to other methylxanthines. In animal studies, high doses of theobromine (Lethal dose 50 in rats 950-1356 mg/kg) exhibited thymic and testicular withering and loss of fetal weight. In human studies, high doses (1-1.5 g/day orally) of theobromine (Lethal dose 50 in humans 1000 mg/kg) showed minor side effects such as nausea, anorexia, sweating, and headache (Monteiro et al. 2016;Salihovic et al. 2014). In the current investigations, we intended to evaluate the neuroprotective properties of theobromine in permanent 2-vessel occlusion (2-VO) rat prototype.
In NSS, each rat was tested for twisting thorax, forelimb flexion, beam-walk, and hanging on a wire, and a cumulative score was calculated individually to present a neurological deficit score (NDS). The sensorimotor performance of rats was assessed on day 1 (before surgery) and days 2, 4, 8, and 12 after occlusion. An accelerating rotarod technique was used to assess the sensorimotor abilities in rats, and fall-off latency was noted (Oyamada et al. 2008). Spatial memory was appraised using the elevated plus-maze test (EPM) (Parle and Dhingra 2003;Itoh et al. 1990). Animals were given acquisition trials on day 12 and retrieval trials on day 13 in EPM, and transfer latency (TL) was recorded. The data of TL were used calculate inflexion ratio (IR): IR = (L 0 -L 1 )/L 0 (L 0 = Initial TL, L 1 = Retention TL). A decrease in IR indicates memory loss and an increase in IR indicates improvement in memory (Rajesh et al. 2017). On day 14, the animals were given trials in a novel object recognition test (NORT) to estimate discrimination index (DI) for evaluation of working memory abilities (Ennaceur 1998;Ennaceur and Delacour 1988).

Biochemical parameters
Animals were sacrificed using the cervical dislocation manner with sodium pentobarbitone (150 mg/kg) anesthesia. The entire brain was dissected out and rinsed in freezing isotonic in dietary cocoa or chocolate products by humans. Data from earlier reports indicate that a 25-225 times increase in plasma theobromine concentrations relative to extreme theobromine intake by humans is devoid of any significant toxic repercussions in rats (Shively et al. 1984). The distribution of rats in 5 diverse groups (n = 6) was accomplished in a single-blind mode using a random distribution method to minimize selection bias. An experimenter blinded to the treatment to be allocated to each animal dispersed the animals into the following groups: (i) Sham-operated (S), (ii) S + T100, (iii) CH, (iv) CH + T50, (v) CH + T100. CH was initiated in rodents on 1st day by performing BCCAO surgery. After 90 min of CCA, ligation rats were subjected to vehicle (0.2% CMC, 5 ml/kg, p.o.) or theobromine (50 and 100 mg/kg, p.o.) treatments that continued for 14 successive days. In sham-operated (S) rats, similar surgery was performed without occluding CCAs and was administered vehicle or theobromine (100 mg/kg) for 14 consecutive days.

Histopathological assessment
Animals were given anesthesia, and reflexes were assessed. Subsequently, intracardiac perfusion through the left ventricle ensued using 10% neutral buffered formalin solution (10% NBF) by means of a gravity-fed perfusion device. Hippocampus and cortical (frontal lobe) regions were secluded and immersed in 10% NBF with 0.05% natriumazid (pH 7.0) (fixative:tissue = 10:1) for 6 days (4 °C). Fixed tissues are kept in 70% ethyl alcohol at 4 °C. Later thin Sect. (5.0 µm) were carved out using a rotary microtome and applied with hematoxylin and eosin (H&E) dye. Permanent slides were scrutinized using light microscopy at × 40 magnifications. In histomorphometric analysis, cortical (frontal lobe pyramidal neurons) and hippocampus (CA 1 and 3) neuron densities (per µm 2 ) were determined by counting viable neurons using ImageJ software (NIH Image 1.61; National Institute of Health; Bethesda, MD).

Statistical analysis
A trained experimenter blinded to drug regimens specified to different cohorts examined and appraised the data. Outliers were absent (Grubb's test) in the data, and the Kolmogorov-Smirnov test and Levene's test established normal distribution of variables and homogeneity of variance (HOV p > 0.05, Levene's test), respectively. Else, in case of unequal variance (HOV p < 0.05, Levene's test), Welch's ANOVA (p < 0.05, F′-statistic) and Games-Howell posthoc tests can be useful. Means of normally distributed variables were scrutinized and related by one-way ANOVA (data from NORT, EPM, and biochemical) or repeated measures two-way ANOVA (data of mNSS and rotarod test). In case ANOVA outcomes are significant (p < 0.05) in F-statistics, multiple comparison tests viz. Tukey's HSD (Honest Significant Difference) or Bonferroni were applied. Data is stated as mean ± Standard Deviation (S.D.). Statistical significance was deemed at p < 0.05. 0.9% w/v sodium chloride solution (sterile). After-homogenate formation (10% w/v phosphate-buffered saline), the samples were centrifugated (CPR-30 Remi Compufuge, Vasai, India) at a 12,000 × g force for 15 min at 4℃. The extra liquid (supernatant) was secluded to estimate biochemical parameters following standard procedures. Thiobarbituric acid reactive substances (TBARS) were assessed by reaction of malondialdehyde in samples with thiobarbituric acid (Ohkawa et al. 1979) that forms a chromophore whose variability in optical density (O.D.) was recorded at 532 nm using twin-beam UV-spectrophotometer. Molar extinction coefficient (ε) = 1.56 × 10 5 /M/cm was applied to compute TBARS stated as nmol per mg protein. In estimating glutathione, Ellman's reagent converts glutathione in the sample to yellow colored 2-nitro-5-thiobenzoic acid whose O.D. variability is noted at 412 nm. Glutathione (µmol GSH per mg protein) was quantified by using ε = 1.36 × 10 4 /M/ cm (Ellman, 1959). Superoxide dismutase (SOD) activity was evaluated by adopting the method of Winterbourn et al. (1975). The SOD in the sample hinders the reduction of nitro blue tetrazolium (NBT) by O 2ˉ and formazan production. The rate of SOD (µmol NBT reduced per min per mg protein) was enumerated using ε (formazan) = 15,000 /M/ cm. Catalase activity (µmol H 2 O 2 decomposed per min per mg protein of brain) was computed using ε = 43.6 /M/cm at λ max = 240 nm (Claiborne 1985). The process elaborated by Sastry et al. (2002) was implemented to assess total nitrites. A typical curve of sodium nitrite (concentration range 0.01-0.1 mM) was designed, nitrite content in samples was equated and then specified as µmol per mg protein. The lactate dehydrogenase activity (µmol NADH oxidized/min/mg protein) was scrutinized by the procedure of Horecker and Kornberg (1948) using ε = 6220 M − 1 cm − 1 at λ max = 340 nm. The acetylcholinesterase (AChE) rate was recorded by the method of Ellman et al. (1961). Rate of AChE (µmol acetylthiocholine (AcTh) iodide hydrolyzed per min per mg protein) was computed utilizing ε = 1.36 × 10 4 /M/cm at λ max = 412 nm. The paper chromatography method was used to assess levels of GABA by using a mobile phase consisting of n-butanol, glacial acetic acid, and water in a ratio of 1:5:10. GABA concentration (pmol/ml) in eluted solution was appraised at 509 nm and by the standard curve of GABA (Swamy et al. 2013). Protein was estimated by the scheme elaborated by Lowry et al. (1951).

Theobromine attenuated memory loss against CH in rats
In the EPM test, TL and IR were estimated to gauge the outcomes of theobromine on spatial memory function of rats against CH. In EPM test acquisition trials, there was no substantial intergroup disparity in day 12 TL [F (4,25) = 0.3212, p < 0.001] (Fig. 3 A). In the retention trials (day 13), a substantial upsurge (p < 0.001) in the TL (Fig. 3B) was pragmatic in response to CH when juxtaposed with vehicle-treated sham-operated rats. Theobromine (50 and 100 mg/kg) administration considerably diminished the CH prompted upsurge in the TL (p < 0.01, p < 0.001) when juxtaposed with CH and vehicle alone treatments [F (4,25) = 120.7, p < 0.001].
In harmony with the results of TL, the IR was significantly reduced (p < 0.001) by CH and vehicle treatments in reference to sham-operation accompanied by vehicle administration ( Fig. 3 C). Theobromine (50 and 100 mg/kg) enhanced (p < 0.05, p < 0.001) the IR against CH in rats when compared to CH and vehicles administered rats [F (4,25) = 113.7, p < 0.001]. Furthermore, appraisal of recognition type memory in NORT by measuring DI on day 14 disclosed waning of working memory owing to permanent 2-VO on the 1st day. CH prompted considerable decline (p < 0.001) in DI in reference to sham-operation [F (4,25) = 26.97, p < 0.001]. Oral post-treatment with theobromine (50 and 100 mg/kg) for 14 days abrogated memory deficits (p < 0.05, p < 0.001) against CH when related with CH and vehicle administrations alone (Fig. 3D). Experimental data showed that theobromine

Theobromine lowered brain acetylcholinesterase activity and upregulated GABA levels against CH
CH initiated considerably (p < 0.001) escalated the rate of brain AChE action and abrogated GABA concentration when juxtaposed to vehicle and sham treatments. A noteworthy decline in rate of AChE (p < 0.05, p < 0.05) [F′ (4,20) = 15.09, p < 0.001] (Fig. 5 A) and improvement in GABA levels (p < 0.05, p < 0.01) [F (4,20) = 17.01, p < 0.001] (Fig. 5B) against CH was pragmatic in response to theobromine (50 and 100 mg/kg) administration when related to CH and vehicle treatments only. These repercussions specified that theobromine post-conditioning considerably attenuated the CH-induced loss of cholinergic and GABAergic neurotransmission in rats.

Theobromine negated neurodegenerative changes in CH rat prototype
In the histopathological investigation, rats subjected to permanent 2-VO showed neurodegenerative changes marked by blebbing of the plasma membrane (b), swelling (s), and pyknosis (p) in the cortex (frontal pyramidal neurons) and Cornu Ammonis 1 and 3 (CA1 and CA3) areas of the hippocampus of the brain. Sham-operation displayed none of the neurodegenerative signs. Theobromine (50 and 100 mg/ kg) abolished neuropathological variations in these brain sub-regions neuronal membrane and chromosomal matter (Fig. 7). Histomorphometric examination disclosed that 2-VO-induced decrease in neuron density was attenuated by theobromine post-treatments with theobromine 100 mg/ kg showing a more pronounced neuroprotective effect when compared to theobromine 50 mg/kg dose (Table 1). Hence, it can be inferred that oral administration of theobromine attenuated CH triggered neurodegeneration in the brain.
The circulatory deviations provoke uninvited proteins (e.g., matrix metalloproteinases) and immunity regulators (e.g., TNF-α, interleukins) that can damage the blood-brain Discussion CH is correlated with neurobehavioral deficits that involve hypoxia and hypo-nutrition-induced pro-oxidants and inflammatory cytokines (Saxena et al. 2015). Reactive oxygen species (ROS) are notorious for promoting endothelial dysfunction and local inflammatory responses via adhesion proteins, phospholipase A 2 , hypoxia-inducible factor-1α, TNF-α, and IL-1β (Chen et al. 2011). ROS modify cellular lipids and proteins to breach cell membrane integrity and cause cell death via genotoxicity (Liu and Zhang 2012). Oxidative decomposition of polyunsaturated fatty acids leads to the formation of a variety of overactive hydroperoxides, radicals, and aldehydes such as hexanal, acrolein, malondialdehyde, propanal, and 4-hydroxy 2-nonenal. These toxic mediators can alter protein structure and lipoproteins (e.g.,  and vasodilatory effects of acetylcholine through nicotinic receptors located on microglia, astrocytes, and blood vessels prevent memory impairments (Maurer and Williams 2017;Jin et al. 2015). Evidence indicates a significant increase in the rate of brain AChE and decline in the rate of choline acetyltransferase (ChAT) action with a concomitant drop in acetylcholine generation lead to memory deficits after CH (Sun et al. 2020;Gnatek et al. 2012). In the recent investigation, CH enhanced the central AChE activity and reduced the GABA content in rats. Reported data suggest that GABAergic depreciation can be a factor for overt glutamatergic excitatory transmission (Asomugha et al. 2010;Liu et al. 2015). GABA is well known for its protective effects (e.g., antioxidative, anti-apoptotic) and has been extensively investigated in pre-clinical studies, confirming its benefits against hyperglycemia, proliferative disorders, liver ailments, kidney disease injury, and neurodegeneration. CBF and energy demand/supply ratio are also improved by GABA (Chen et al. 2019;Ngo and Vo 2019).
Neuronal hyperpolarization reduces metabolic activity, ROS, and inflammation, parallel to hypothermia (Lee et al. 2018;Neumann et al. 2013). Hence, in excitotoxicity origin brain dysfunction, GABAergic hyperpolarization can afford great relief. Theobromine was able to diminish the brain AChE activity and augment the GABA levels against CH. These findings corroborated that theobromine can increase cholinergic transmission and deter excitotoxic pathways by enhancing the GABA levels in CH states. Pre-existing cardiovascular and metabolic disorders cause hemodynamic changes in the whole brain that forms the basis of CH (Traystman 2003), which can be closely simulated by the permanent BCCAO technique in rodents (Bacigaluppi et al. 2010). 2-VO is a forebrain ischemia model that can be divided into acute (24 h), subacute (3 days), and chronic phases (> 7 days) (Ma et al. 2020) in which hippocampal CA1 neurons are the most vulnerable and cortical (including neocortex) are late ischemic tolerant followed by subcortical structures such as caudateputamen, striatum, and thalamus (Hossmann 2008). In the present study, CH produced substantial neurodegenerative deviations noticeable by pyknosis, swelling, and blebbing of membranes in the hippocampus (CA1 and CA3 regions) and cortical regions, and these changes were markedly attenuated by theobromine post-treatment in rats. Hence, the histopathological investigation data reinforced the present biochemical outcomes.
The present research assessed neurological, sensorimotor, and memory functions at different time intervals starting from 1st day. Results showed that permanent 2-VO produced CH that caused a significant increase in their neurological scores indicating deficits in balance, gait, sensory functions, and reflexes, and a decrease in motor activity measured over barrier and other cerebral capillary networks ). Subsequently, migration of plasma leucocytes in the brain parenchyma, invasion of neurotoxins, and glial activation perpetuate inflammatory cascade. An upsurge in IL-1β levels early during hypoperfusion is implicated in activating inflammatory intermediaries viz. phospholipase A 2 , cyclooxygenase-2, and iNOS (Woodcock and Morganti-Kossmann 2013). An increase in IL-6 levels is also linked with brain infarct, thrombus formation, and an upsurge in TNFα and ROS yield through microglia and astrocytes (Tang et al. 2015). TNF-α stimulates caspase-initiated apoptotic mechanisms through TNFR1 receptors, necrosis (via excitotoxic and nitric oxide pathways) and transcription activity of NF-κB substantially (Duncan et al. 2020;Ju Hwang et al., 2019). These proceedings deteriorate capillaries, tight junction proteins, and extracellular matrix, ensuing detrimental consequences such as white matter atrophy and loss of neurobehavioral functions Fogal and Hewett 2008;Maher et al. 2003). In the existing investigation, CH substantially amplified the content of pro-inflammatory biomolecules (TNF-α, IL-1β, IL-6), which materialize their neurotoxic repercussions from the sub-acute period of CH and onwards. Furthermore, the activity of LDH and caspase-3 levels (cell death markers) was also enhanced by CH over 14 days. Oral administration of theobromine for 14 days abolished the TNF-α, IL-1β, IL-6, caspase-3 levels, and LDH activity in the brain of rats subjected to CH. Inflammatory cascade, the appearance of cytokines/chemokines, and apoptosis machinery are regulated by NF-κB, whose transcription activity is amplified when there is a drop in CBF (Saggu et al. 2016). Brain insult in the form of traumatic brain injury (TBI), CH, excitotoxic pathways, and several neurotoxins (free radicals) (Negre-Salvayre et al. 2008) can galvanize the NF-κB action resulting in an upsurge in cell degrading enzymes (e.g., matrix metalloproteinases), cytokines (e.g., IL-6, C-reactive protein), and inflammatory molecules (e.g., selectins, integrins, iNOS, cyclooxygenase-2) (Liu et al. 2017;Duncombe et al. 2017). In harmony with previous conclusions , CH significantly enhanced the brain in the existing pre-clinical investigation. Treatment with theobromine (100 mg/kg) attenuated the CH-induced brain NF-κB levels that might have contributed to lowering inflammatory cytokines (TNF-α, IL-1β, IL-6). These findings disclosed that theobromine declined the CH triggered inflammation and secondary brain damage in rats.
Funding The present research received no specific funding.

Availability of data and material
The data used to support the findings of this study can be made available upon a reasonable request to the corresponding author.

Conflict of interest None declared.
Ethics approval The research protocol was approved by Institutional Animal Ethics Committee vide approval reference no. SSP/ IAEC/2019/009 on date: 17-11-2019. Animals were housed within the institutional establishment (Animal House Facility) registered under CPCSEA (Regd. 1616/PO/Re/S/12/CPCSEA). All the animal tests were performed following the guiding principles of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), Ministry of Environment and Forests (Animal Welfare), Government of India (GOI), New Delhi.

Consent to participate Not applicable.
Consent for publication It is affirmed that all the authors have seen and agreed to the submission and publication of the research article and their inclusion of name(s) as co-author(s). 14 days duration. Findings from previous studies suggest that methylxanthines such as pentoxifylline (Dong et al. 2018;Eun et al. 2000) and caffeine (Rehni et al. 2007;Bona et al. 1995) can defend against hypoxia-ischemia conditions (Cova et al. 2019;Kumral et al. 2010) via mechanisms linked to phosphodiesterase-4 and adenosine receptors inhibition. In healthy elderly humans, flavanol-rich cocoa improved CBF in the middle cerebral artery, which substantiates that cocoa and its constituents may benefit cerebral ischemia (Sorond et al. 2008). Parallel to these findings (Camandola et al. 2019;Onatibia-Astibia et al., 2017), current results indicated that theobromine, when given orally for two weeks, improved the neurological and sensorimotor abilities in rats marked by a decrease in NDS and upsurge in fall-off latency respectively. In the present study, theobromine significantly decreased the TL (day 13). It enhanced the IR (day 13) and DI (day 14), which indicated improved spatial and recognition-type working memory in rats against CH. Precise coordination between different brain structures such as the cortex, thalamus, hippocampus, amygdala, limbic system, medulla, and cerebellum regulates spatial orientation, awareness, recognition-type memory, balance, motor coordination, reflexes, sensory functions, and gait (Ackerman 1992). The biochemical analysis in the entire brain disclosed a decline in oxidative stress, inflammation, and cell death biomarkers and an increase in neurotransmitters, which aptly substantiated the behavioral findings in the present study. Commensurate with earlier findings, hippocampus and cortical tissues are the most vulnerable regions in BCCAO induced CH model supported by the H&E staining technique in this study. Besides, we observed a dose-dependent amelioration of biochemical outcomes and neurobehavioral functions in animals by theobromine.

Conclusions
In this study, the anti-oxidative and anti-inflammatory effects of theobromine abolished CH-associated cell death pathways with a concomitant upsurge in neurotransmitters (GABA) and decline in acetylcholinesterase activity, thereby resulting in improvement of neurological, sensorimotor, and memory functions in rats in the CH model. These are novel research findings indicating that theobromine can be used against hypoperfusion-associated cerebral disorders such as Alzheimer's disease, vascular cognitive impairment, and dementia.