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 A2, 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., low-density lipoproteins) of physiological and structural importance (Negre-Salvayre et al., 2008). Furthermore, CH triggers nitrosative stress through neutrophils, macrophages, and microglia-associated inducible nitric oxide synthase (iNOS). Peroxynitrites modify cell proteins (tyrosine nitrosylation, carbonylation) and DNA causing irreversible cell injury, necrosis, and microvascular abnormalities (Daulatzai, 2017). Nitric oxide-dependent attenuation of respiratory complex I and II and instigation of poly(ADP-ribose) polymerase accentuate ROS-associated neurodegeneration. In the CH state, depletion of endogenous antioxidants augments the rate of free-radical yield and reactive, secondary intermediaries of oxidative insult. Studies on transgenic animals suggested that SOD and catalase actively confer neuroprotection against the decrease in CBF (Warner et al., 2004). In existing experiments, CH significantly augmented the TBARS in the brain homogenate samples and total nitrites, and both of these reactive by-products were abrogated by theobromine. TBARS directly specifies malondialdehyde (MDA), which happens to be notorious lipid peroxidation aldehyde, accountable for several biochemical aberrations (Ayala et al., 2014). Along with a decrease in lipid peroxidation, theobromine augmented endogenous brain antioxidants (GSH, SOD, and catalase) in the CH rat prototype.
The circulatory deviations provoke uninvited proteins (e.g., matrix metalloproteinases) and immunity regulators (e.g., TNF-α, interleukins) that can damage the blood-brain barrier and other cerebral capillary networks (Wang et al., 2020). 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 A2, 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 TNF-α 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 (Wang et al., 2020; 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 (Li et al., 2020), 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.
Central cholinergic transmission regulates cognitive functions, several biological activities (e.g., anti-apoptotic factors, stress response, wakefulness, sleep), and the release of neuromodulators (e.g., dopamine, growth factors) (Resende and Adhikari, 2009). Published data indicate that acetylcholine modulates several stages of cognitive processing (Ferreira-Vieira et al., 2016). Anti-inflammatory 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 caudate-putamen, 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 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.