[1] 16w Neurotoxic effects of acyclovir: impacts on oxidative stress, inflammation, and neurotransmitter dynamics in male Wistar rats

[1] 233w Background Acyclovir is a potent antiviral agent with variable side effects on the central nervous system. Although previous studies have shown that acyclovir has neurotoxic effects, there is a dearth of scientific information on the mechanisms through which acyclovir induces neurotoxicity. Aim Thus, the present study assessed the impact of acyclovir on oxidative stress, inflammatory markers, and neurotransmitter levels in the cerebellum, prefrontal cortex, and basal ganglia and its potential impact on cognition and motor function. Materials and methods Twenty-eight male Wistar rats (120-150 g) were randomly assigned into four equal groups. The control group received distilled water while acyclovir-treated groups received single daily treatment at doses of 10, 20, and 40 mg/kg bw orally for 28 days.Results Acyclovir, at 20 and 40 mg/kg but not at 10 mg/kg, induced a decline in memory, spatial learning, and motor coordination when compared with the control. Also, the brain levels of antioxidants (catalase, superoxide dismutase, and glutathione) and anti-inflammatory cytokine IL-10 were significantly reduced while malondialdehyde and pro-inflammatory cytokines (IL-6 and TNF-α) were increased in the cerebellum, prefrontal cortex, and basal ganglia in acyclovir-treated rats, particularly in those treated with 20 and 40 mg/kg. Serotonin levels increased while dopamine levels decreased in the brain tissues of acyclovir-treated rats. However, IL-1β was not significantly affected. Conclusion Acyclovir impaired motor function, muscle strength, and memory by inducing derangement of the brain's oxidative markers, cytokines, and neurotransmitter levels.

[1] 433w Antiviral agents are useful in the therapeutic management of viral infections, providing effective treatment alternatives for a multitude of patients globally. The administration of antiviral agents is not devoid of risks, as prior studies indicate their potential association with toxic effects (Akhigbe et al., 2021;Akhigbe et al. 2024a), including neurotoxic side effects (Bertrand et al. 2021). These neurotoxic effects present a broad spectrum of severity, ranging from mild cognitive dysfunction and alterations in mood to more severe conditions such as seizures, neuropsychiatric disturbances, and in certain instances, life-threatening encephalopathies (Zareifopoulos et al. 2020). The underlying mechanisms contributing to the neurotoxicity of antiviral agents are complex, varying with the specific pharmacodynamic properties of the antiviral agent used (Lanman et al. 2021). Also, individual patient health status can modulate both OO Obembe olawale.obembe@uniosun.edu.ng RE Akhigbe reakhigbe@lautech.edu.ng the probability and intensity of these neurotoxic outcomes. Numerous established pathways have been identified concerning antiviral-induced neurotoxicity and that of their metabolites. This includes immune-mediated responses, wherein the pharmacological agent incites an aberrant immune reaction within the central nervous system (Jafari et al. 2014). Mitochondrial dysfunction and oxidative stress represent another mechanism whereby antivirals disrupt cellular bioenergetics and augment the production of deleterious free radicals (Kaur et al. 2023). Pathological alterations in neurotransmitter homeostasis can affect normal cerebral function, resulting in a variety of neurological and psychological manifestations (Picca et al. 2020;Teleanu et al. 2022). Furthermore, the accumulation of these pharmacological agents and their metabolites within the CNS can result in direct neuronal damage. The blood-brain barrier (BBB) is traditionally regarded as a protective structure that safeguards the brain from potentially deleterious substances present in the systemic circulation. Nonetheless, in circumstances characterized by inflammation or compromised immune systems, antivirals and their metabolites may pass through this protective barrier with ease, causing CNS toxicity (Calcagno et al. 2014). The increased permeability of the BBB under specific pathological conditions accounts for the differential susceptibility of certain patients to antiviralinduced neurotoxicity (Archie et al. 2021). The risk for neurotoxic effects associated with antiviral agents is influenced by a multitude of factors. These encompass the pharmacological dosage, with elevated doses typically correlating with an increased risk of adverse effects. The duration of treatment is also a determining factor, as extended exposure may result in cumulative neurotoxic consequences. The method of drug administration can influence the rate and concentration at which the pharmacological agent penetrates the CNS. Additionally, the overall health status of the individual receiving the treatment, including variables such as age, renal function, and the presence of comorbid conditions, can significantly affect the likelihood of encountering neurotoxic effects (Robertson et al. 2012).

[2] 370w Acyclovir (9-[2-hydroxymethyl] guanine) is an analog of nucleoside that is used in the management of varicellazoster virus and herpes simplex virus types 1 and 2 (HSV-1, HSV-2) infections. Its mechanism of action involves the inhibition of viral replication. Following cellular uptake, acyclovir is enzymatically transformed into acyclovir monophosphate by the virus-specific thymidine kinase. Subsequently, intracellular enzymes phosphorylate it further to acyclovir triphosphate, which exerts competitive inhibition on viral DNA polymerase, consequently arresting viral replication (Majewska and Mlynarczyk-Bonikowska 2022). While Zachary et al. (2008) suggested that acyclovir demonstrates a certain degree of drug specificity, with minimal impact on uninfected cells, multiple studies have raised concerns regarding its toxicological potential. Investigations by Movahed et al. (2013) and Patel et al. (2019) report various forms of toxicity associated with acyclovir administration. Studies show that acyclovir-induced neurotoxicity primarily occurs in individuals with advanced age or renal impairment (Patel et al. 2019;Brandariz-Nuñez et al. 2021). Additionally, Ernst and Franey (1998) noted that neurotoxicity was more prevalent in immunocompromised patients or those receiving high doses of acyclovir. These findings highlight the necessity for further research into the safety profile of acyclovir, particularly in healthy subjects. Given the widespread use of acyclovir and the potential neurotoxic effects associated with its administration, there is a pressing need for in-depth research into its impact on central nervous system function. Although the brain has several parts, the prefrontal cortex, cerebellum, and basal ganglia play key roles in motor function. In addition, the prefrontal cortex plays a role in attention, working memory, decision-making, and behavioral control, while the cerebellum is essential for fine-tuning motor activity, balance, and coordination, and the basal ganglia are key regulators of motor control, motivation, and reward processing (Ajayi and Akhigbe 2021). Thus, assessment of these areas of the brain provides necessary information on the processes they regulate. Therefore, the current study investigated the effects of acyclovir administration on various aspects of neuronal function and histoarchitecture integrity in adult male Wistar rats. Additionally, its potential effects on cognitive functions, such as learning and memory, as well as motor functionality were assessed. Specifically, its impacts on oxidative stress parameters, inflammatory pathways, and neurotransmitter concentrations in key brain regions, including the cerebellum, prefrontal cortex, and basal ganglia were explored.

[1] 135w In the cerebellum, administration of acyclovir (20 and 40 mg/kg) caused a significant decrease in the activities of catalase and SOD, and GSH levels but an increase in MDA when compared with the control group. The apparent alterations in the markers of oxidative stress at 10 mg/kg dose was not statistically significant. Also, in the prefrontal cortex, acyclovir at 40 mg/kg, but not at 10 and 20 mg/kg, significantly reduced catalase activities and GSH levels. Interestingly, SOD activity in the prefrontal cortex was comparable across the groups. In the basal ganglia, acyclovir (10, 20, and 40 mg/kg) decreased the activities of catalase and SOD, and GSH levels when compared with the control group. However, MDA was significantly increased in the prefrontal cortex and basal ganglia at 10, 20, and 40 mg/kg of acyclovir (Fig. 3).

[2] 146w Acyclovir (20 and 40 mg/kg) caused a significant decrease in the time spent by Wistar rats exploring the novel object when compared with the control and acyclovir-10 mg/kg-treated rats, indicating a decline in short term memory caused by 20 and 40 mg/kg acyclovir. The observed decrease in time spent exploring the novel object was significantly more at 40 mg/kg dose than at 20 mg/kg dose, while 10 mg/kg had no significant effect. Likewise, acyclovir (20 and 40 mg/kg) caused a significant increase in the escape latency period of Wistar rats in the Morris water maze test when compared kg dose respectively. Acyclovir decreased short term memory (exploration time) and increased escape latency, indicating decline in spatial memory prefrontal cortex and basal ganglia when compared with the control. Conversely, dopamine levels decreased in these brain regions in acyclovir treated rats when compared with the control (Fig. 5).

[3] 133w Administration of 10, 20, and 40 mg/kg of acyclovir led to a significant rise in the serotonin levels in the cerebellum, Fig. 4 Effect of varying doses of acyclovir on inflammatory cytokines in selected parts of the brain in male Wistar rats. Values are Mean ± SEM of 7 replicates, p < 0.05. a, b -indicates significant dif-ference from control and 10 mg/kg dose respectively. A dose related increase in pro-inflammatory cytokines and decrease in anti-inflammatory cytokines were observed 1 3 indicating astrogliosis. The astrocytes appear hypertrophied and increased in number with several dark, dense clusters that could represent cellular aggregates, which might be microglial nodules or potentially even amyloid plaques. The distribution of cells and cellular processes appears uneven and disorganized with irregular shapes and sizes and degeneration (Figs. 6 and 7).

[1] 462w Acyclovir is a highly effective antiviral drug with an acclaimed safety profile. Its antiviral potency against the herpes virus is attributed to its viral DNA synthesis inhibition (Stahl and Mailles 2019). Although studies have shown that acyclovir exhibits cell-specific antiviral activity with minimal cytotoxicity on uninfected cells, concerns have been raised regarding its toxicology. Notably, advanced age normally distributed neuronal and glial cell populations and there were no abnormalities observed. However, neuronal architecture was disrupted, with neuronal loss and damage in acyclovir (10 and 20 mg/kg) treated rats. Also, neuropil and glial process alterations were observed. Acyclovir (40 mg/kg) had a more severe impact on the cortical tissue, causing a severe reduction in the pyramidal cell population with a consequential increase in the population of the astrocytes and an abundance of dark reactive astrocytes showing astrocytic hypertrophy. The basal ganglia of control rats had a normally distributed neuronal and glial cell population. The histoarchitecture of acyclovir (10 mg/kg) rats also showed no abnormality. However, acyclovir (20 mg/ kg) treated rats had basal ganglia with mild neuronal loss when compared to the control group but had a fairly normal distribution of neuronal and glial cells. The basal ganglia of acyclovir (40 mg/kg) showed an abundance of star-shaped cells with multiple processes. These are reactive astrocytes, Fig. 5 Effect of varying doses of acyclovir on serotonin and dopamine levels in selected parts of the brain in male Wistar rats. Values are mean ± sem of 7 replicates, p < 0.05. a, b,c -indicates significant differ-ence from control, 10 mg/kg dose and 20 mg/kg dose respectively. Serotonin levels increased while dopamine levels decreased in acyclovir treated rats the potential mechanisms through which it induces neurotoxicity. Thus, the effects of acyclovir administration on oxidative stress, inflammation, neurotransmitters, and histoarchitecture of the cerebellum, prefrontal cortex, and basal ganglia were investigated in this study. The results of and renal dysfunction (Patel et al. 2019;Brandariz-Nunez et al., 2021) and immunosuppression or high doses (Ernst and Franey 1998) have been identified as risk factors for acyclovir-induced neurotoxicity. However, there is a dearth of information on the neurotoxic effects of acyclovir and ous neuronal degeneration and loss. Red arrow indicates alterations in neuropil and glial processes; d (40 mg/kg acyclovir): shows cortical tissue with severe reduction in the pyramidal cells and gliosis, arrow indicates hypertrophy of astrocytes (thin yellow arrow) iii. a: normal basal ganglia with properly distributed neuronal and glial cells; b (10 mg/kg acyclovir): neurons are normal and no abnormality was observed (black arrow); c (20 mg/kg acyclovir): fairly normal distribution of neuronal and glial cells; d (40 mg/kg acyclovir): arrow indicates abundance of star-shaped cells with multiple processes indicating astrogliosis. The astrocytes appear hypertrophied and cells are disorganized with irregular shapes and sizes with imminent degeneration (blue arrow)

[2] 39w living organisms. An imbalance between ROS production and antioxidant defenses triggers oxidative stress, causing damage to proteins, nucleic acids, and lipids thus disrupting normal physiological processes, which can lead to the development of neurodegenerative disease (Feng et al. 2022).

[3] 179w The present results showed that the administration of acyclovir caused a significant decrease in the activities of these antioxidants across the three brain regions. The decrease in the antioxidant activities and levels suggests an overwhelming increase in the production of free radicals and a breach in the antioxidant defense system (Hopps et al. 2010). Acyclovir might have decreased the total brain antioxidants level by generating reactive singlet and triplet oxygen species, thereby causing the induction of oxidative stress (Federico et al. 2012;Gbadamosi et al. 2016). Acyclovir neurotoxicity may have been mediated, according to this finding, by augmenting the interaction of ROS with this study showed that the administration of acyclovir led to an increase in the inflammatory response in the brain, which caused a reduction in the activities and levels of the antioxidants and promoted oxidative stress in the cerebellum, prefrontal cortex, and basal ganglia, and pathological degenerations in their histoarchitecture. These culminated in a negative impact on their physiological functions viz. motor activity and memory. These findings demonstrate that acyclovir induces detrimental effects on cognition and locomotive activities.

[4] 193w To elucidate the possible mechanisms by which acyclovir induced derangement of cognition and locomotion, the effects of acyclovir on the activities of catalase and superoxide dismutase, and glutathione levels, which are at the forefront of the oxidative defense system were evaluated in the brain. Antioxidants are compounds that inhibit oxidation as a reducing agent by scavenging reactive oxygen species in Fig. 7 Effect of varying doses of acyclovir on neuronal density and neuronal length in selected parts of the brain in male Wistar rats. Values are expressed as Mean ± SEM of 3 replicates, p < 0.05. a, b,c -indi-cates significant difference from control, 10 mg/kg dose and 20 mg/ kg dose respectively. Neuronal density and neuronal length of brain regions decreased in acyclovir treated rats Therefore, acyclovir-induced elevated pro-inflammatory cytokines and reduced anti-inflammatory cytokines in this study contributed, at least in part, to the observed histopathological alterations and neurobehavioral changes. The decline in motor and memory functions and histopathological changes observed in the brain following acyclovir therapy can be linked to the noticeable neuroinflammation in the cerebellum, prefrontal cortex, and basal ganglia (Agusti et al. 2017;Cabrera-Pastor et al. 2019;Liu et al. 2022).

[5] 193w Dopamine and serotonin are crucial neurotransmitters regulating various neurological functions. Dopamine controls reward, cognition, and movement while serotonin influences mood, cognition, and other processes (Torrisi et al. 2020;Franco et al. 2021;Akhigbe et al. 2023;Besong et al. 2023). These neurotransmitters are nontoxic at physiological levels, but in excess or at suboptimal levels, they may impair neuronal functions, and their physiological functions are lost. The alterations in the levels of these neurotransmitters following acyclovir administration may also explain the impaired motor function and memory deficit. It is also possible that the release of pro-inflammatory cytokines and oxidative stress contribute to the downregulation of the expression of tyrosine hydrolase and the upregulation of monoamine oxidase leading to the excessive breakdown of dopamine (Ostadkarampour and Putnins 2021), further contributing to observed decline in dopamine concentrations in acyclovir-treated rats. Also, the crosstalk between neurotransmitter regulations may be impaired by neuroinflammation and neuronal damage and the loss of the inhibitory feedback between the neurotransmitters could lead to an increase in serotonin levels (Abg Abd Wahab et al. 2019;Teleanu et al. 2022). Thus, acyclovir-induced neurotoxicity and deficit in motor and cognitive functions may involve multiple pathways; oxido-inflammation and neurotransmitter modulation.

[6] 383w The observed neurobehavioural pattern in acyclovirtreated rats, especially those treated with (20 and 40 mg/ kg, demonstrate that acyclovir-induced cognitive decline evidenced by a decline in memory and spatial learning, and impaired motor function evidenced by a noticeable decline in motor coordination and muscle strength. These observations are due, at least in part, to acyclovir-induced oxidative stress within the brain, leading to neuronal damage and impaired cognitive and motor functions. The brain is susceptible to oxidative stress-mediated injury because of its high metabolic rate, iron-rich areas, and lipid-rich neuronal membranes (Hassan et al. 2022). Oxidative stress induces neuronal mitochondrial dysfunction and destruction of neuronal membrane, leading to accelerated neuron degeneration, impaired signaling, and a decline in motor coordination and cognitive function (Wang et al. 2022). Acyclovirinduced cytokine upregulation also likely contributed to major cellular macromolecules to cause oxidative modification (as seen in the levels of MDA) and to a degree overburden and weakening of the natural antioxidant defence system (Habtemariam 2017). This cascade of events may explain the noticeable histopathological lesions and neurodegeneration (Omotosho et al., 2018). It is also likely that acyclovir-induced ROS-driven oxidative stress is accompanied by impaired oxidative phosphorylation and decreased ATP production (Papa et al. 2012), leading to an increased electron leakage from the electron transport chain, which in turn interacted with the formed ROS to exacerbate the oxidative damage and culminate in the observed histopathological lesions and neurodegeneration with consequent decline in motor function and memory (Schwabe et al. 2012;Nassiri-Asl et al. 2013;Shi et al. 2014). Antioxidants are vital for maintaining redox balance in the brain by neutralizing reactive species and preventing oxidative damage (Famurewa et al. 2025). In this study, acyclovir administration at 20 and 40 mg/kg resulted in a significant reduction in antioxidant enzyme activities (SOD, CAT, and GSH) across all examined brain regions. This decline suggests that the brain's antioxidant defense system was compromised. Importantly, malondialdehyde (MDA), a well-established marker of lipid peroxidation and oxidative damage, was significantly elevated in the same brain regions. The increase in MDA levels supports the occurrence of oxidative stress and membrane lipid damage, even though ROS themselves were not directly measured. The combination of elevated MDA and reduced antioxidant enzyme activity reasonably indicates that oxidative injury occurred, likely as a result of increased ROS generation secondary to acyclovir exposure.

[7] 175w To further elucidate the underlying mechanisms of acyclovir action, we further sought to determine the impact of acyclovir on the expression of brain cytokines. Interleukins are a group of inflammatory cytokines that have a variety of physiological roles as well as pathological implications in both health and diseases. The present findings showed that acyclovir (20 and 40 mg/kg) caused a significant increase in the levels of proinflammatory cytokines IL-6 and TNF-α and a decrease in anti-inflammatory cytokine IL-10 in the three brain regions examined, indicating brain inflammatory injury. IL-6 plays diverse roles in mediating inflammation, neurogenesis, and cell survival (Johnson et al. 2020;Kang and Kishimoto 2021;Rose-John et al. 2017) and a rise in IL-6 is associated with brain damage and neurological disorders (Velliereset al., 2002) as well as memory and spatial learning impairment by activating NADPH-oxidase (Dugan et al. 2009). Also, TNF-α is involved in innate immune response and promotes inflammation, and IL-6 activates TNF-α (Clark 2007), while IL-10 regulates immune homeostasis and attenuates inflammation, thus preventing tissue damage (Saraiva and O'Garra 2010;Yeunget al. 2023).

[8] 305w 1 3 may not directly depict ROS concentration, it indicates the level of ROS produced. While this study did not directly measure reactive oxygen species, the marked increase in MDA levels along with the suppression of key antioxidant enzymes suggests that oxidative stress played a central role in acyclovir-induced neurotoxicity. It is plausible that prolonged acyclovir exposure led to redox imbalance and promoted oxidative damage to neuronal lipids and proteins. This oxidative burden may be responsible for the histological alterations and behavioral impairments observed, including deficits in memory, learning, and motor coordination. Therefore, the findings implicate MDA accumulation and antioxidant depletion as key mechanisms in the neurotoxic profile of acyclovir, with ROS generation inferred as a likely contributing factor. Although biochemical markers were measured, the study did not delve into detailed mechanistic analyses of how acyclovir affects neuronal signaling pathways, such as acetylcholinergic, glutamic, and nitric oxide signaling pathways. This limits the understanding of the specific molecular mechanisms involved in acyclovirinduced neurotoxicity. This study did not also explore the histopathological and biochemical alterations in the hippocampus, a region of the brain that also influences cognition; thus, limits the depth of our findings. Furthermore, the 28-day therapy period may not fully capture the long-term effects of acyclovir on neurobehavioral outcomes. Chronic the observed motor and cognitive deficit. These cytokines possibly induce inflammation, leading to increased bloodbrain-barrier permeability, accumulation of inflammatory mediators in the brain, local inflammatory response, and neuronal injury (Liesz and Kleinschnitz 2015). This leads to loss of neurons and synaptic connections that manifest as cognitive and motor deficits (Liesz and Kleinschnitz 2015). Neuro-inflammation also alters neuronal function, neurotransmission, and synaptic plasticity; thus, impairing motor function and cognition (Lasisi-Sholola et al. 2024). Additionally, alterations in serotonin and dopamine levels might affect neuronal communication and ultimately impair cognitive and motor functions (Di Giovanni et al. 2010).

[9] 168w The present study demonstrates several important findings, but it also has some limitations. The use of only 28 male Wistar rats may limit the statistical power and generalizability of the present findings. A larger sample size could provide more robust data and support sturdier conclusions. Also, this study focused solely on male rats, which might limit the applicability of the results to female populations. Gender differences in pharmacokinetics and neurobiology may influence the effects of acyclovir, suggesting the need for further studies including both sexes. Furthermore, ROS generation was not directly assayed but MDA, the outcome of lipid peroxidation, was determined as an index of ROS-driven lipid peroxidation; although this Fig. 8 Schematic illustration of the effect of acyclovir on neurocognition and motor function exposure and the resulting cumulative effects over longer periods may yield different results. More so, this study did not assess whether the neurobehavioral impairments were reversible after the cessation of acyclovir treatment. Follow-up studies might be necessary to determine if recovery occurs over time.

[1] 110w Overall, the study concludes that acyclovir-induced neurotoxicity may arise from a multifaceted mechanism involving oxidative injury, neuroinflammation, and neurotransmitter dysregulation, leading to adverse neurobehavioral outcomes (motor and cognition deficits) in the treated rats (Fig. 8). However, future studies with larger sample sizes and comparison of responses in both genders for longer periods are recommended. This would provide more robust data and support stronger conclusions. Studies evaluating whether or not acyclovir-induced cognitive deficits are reversible are crucial to determining the long-term effect of the drug. More so, exploration of other neuronal signaling pathways in acyclovir therapy is necessary to deepen the understanding of the specific molecular mechanisms involved in acyclovir-induced neurotoxicity.

[1] 87w Short-term memory was assessed by Novel Object Recognition (NOR) test using the method described by Asif-Malik et al. (2017) and Lasisi-Sholola et al. (2024), with a onehour difference between the familiarization and test phases. Using an 80 × 60 × 40 cm box, the rat explored two familiar objects (FO 1 and FO 2 ) for five minutes (T1). Then after an hour delay, FO 2 was replaced with a new object (NO) for five minutes (T2). The exploration time of FO 1 and NO was determined.

[2] 99w The Morris Water Maze was used to assess spatial learning and memory as reported by Nunez (2008). A 100 cm diameter and 30 cm deep pool with an escape platform was used. After 24 h training where the rats learned to find the platform within 15 s, the pool was coloured with milk and the escape latency was measured as the time to find the hidden platform. A short training session was allowed to avoid stress and minimize the potential for distress associated with repeated exposure to novel stimuli and assess shortterm spatial memory (D'Hooge and De Deyn 2001).

[3] 109w Motor balance was measured according to the study of Goldstein and Davis (1990) using the modified beam-walking. The beam-walk performance was evaluated by letting individual rats walk across a surface of 2.5 cm x 112 cm (width x length), elevated 60 cm above ground level. The beam was marked at intervals, allowing for precise measurement of performance as individual rats walked across. Also, a 20 × 25 × 24 cm box with a 10 cm opening was placed at one end of the beam. The time taken for the rats to transverse the beam and enter the dark box was recorded, and this was repeated twice during each trial.

[4] 84w Muscular strength endurance of the hind limbs was determined as described by Chen et al. (2019) with some modifications. Rats were taken up and supported by their trunks before being permitted to grasp a hanging steel wire (35 cm long, 2 mm diameter, 50 cm above the surface) using their forepaws. The latency to fall from the wire onto the soft flat pad was measured. For each rat, the trial was carried out three times and the longest duration value was used for evaluation.

[5] 29w Serotonin (catalog number: MB-3179 A) and dopamine (catalog number: MB-3092 A) were measured using their respective ELISA kits following the manufacturer's instructions (Nanjing Mornmed Medical Equipment Co., Ltd, China).

[6] 30w with the control and acyclovir-10 mg/kg-treated rats, indicating a decline in spatial memory. The escape latency was also significantly more at 40 mg/kg dose than at 20 mg/kg (Fig. 1).

[7] 62w The 10% neutral buffered formalin-fixed tissues were embedded in paraffin, then 5 μm thick sections were obtained and stained with the Golgi silver staining. The slides were viewed under a light microscope (Celestron Lcd Digital Microscope, Model 44348) and photomicrographs were taken (200 x). Photomicrographs were imported onto image J for quantification as earlier reported (Akhigbe et al. 2024b;Lasisi-Sholola et al. 2024).

[8] 44w Statistical analysis was done using GraphPad Prism version 8.0.2. The differences between groups were analyzed by one-way ANOVA and Bonferoni post-hoc test, and the differences were considered significant at p < 0.05. Values are shown as mean ± standard error of the mean (SEM).

[1] 119w Twenty-eight adult male Wistar rats (10-12 weeks old), weighing between 160 and 180 g, were used for this study. The animals were housed in plastic cages with wired covers in the Animal House of the Department of Physiology, Osun State University, Nigeria. The rats had unrestricted access to rat feed and clean water. The guidelines of the National Research Council (2011) were followed in the handling and care of the animals. After an acclimatization period of 2 weeks, the rats were randomly assigned into four equal groups (n = 7 rats per group). Group A was the control and received 0.2 ml distilled water as a vehicle, while Groups B -D received 10, 20, and 40 mg/kg acyclovir respectively.

[2] 130w Treatments were once daily and via oral route using an oral cannula (18G) for 28 days. The dose of 40 mg/kg/day was obtained from the dose-response curve of our pilot study, which is equivalent to the human dose of 400 mg/day in an average adult. A low dose (10 mg/kg) and medium dose (20 mg/kg) were obtained as 25% and 50% of the actual dose. Although acyclovir is commonly used for 10 days, it is used for 28 days or more to prevent HSV and varicellazoster virus (VZV) reactivation, and in the treatment of shingles, HSV encephalitis, neonatal HSV, and chronic suppressive therapy for recurrent genital herpes (Szenborn et al. 2016;Kłysik et al. 2020;Wilms et al. 2022). Hence, the present study evaluated the impact of acyclovir use for 28 days.

[3] 115w All rats were sacrificed 24 h after the last treatment by euthanasia (30 mg/kg sodium pentobarbital, intraperitoneal). They were carefully decapitated and different regions of the brain viz. the cerebellum, prefrontal cortex, and basal ganglia were dissected. The right hemisphere of the harvested organs was weighed and homogenized in a phosphate buffer solution (Na 2 HPO 4 and NaH 2 PO 4 pH 7.4, 0.1 M). The resulting homogenates were centrifuged in a cold centrifuge at 4 °C for 10 min at 10,000 rpm, and then the supernatant was collected and preserved at -20 • C for biochemical assays. The left hemisphere of these organs was fixed in 10% neutral buffered formalin for histopathological examinations.

[4] 57w Malondialdehyde (MDA), Catalase, superoxide dismutase (SOD), and reduced glutathione (GSH) were assayed by colorimetry. Catalase activity was determined as reported by Sinha (1972), MDA was assayed as reported by Lasisi-Sholola et al. (2024), while SOD activities were determined as described by Misra and Fridovich (1972), and GSH levels were assayed as reported by Sedlak and Lindsay, (1974).

[5] 47w The levels of interleukin-1β (IL-1β) catalog number: 22599 A), IL-10 (catalog number: MB-25369 A), IL-6 (catalog number: MB-2899 A), and tumor necrosis factor-α (TNF-α) (catalog number: MB-2868 A), were determined using their respective ELISA kits according to the manufacturer's instructions (Nanjing Mornmed Medical Equipment Co., Ltd, China).

[6] 88w Using beam cross time as an index of motor balance, acyclovir treatment (20 and 40 mg/kg) resulted in a significant increase in the beam cross time of Wistar rats when compared with the control group. The recorded increase in beam cross time was significantly higher at 40 mg/kg than at 20 mg/kg. More so, acyclovir at 40 mg/kg, but not at 10 and 20 mg/kg, resulted in a significant decrease in grip hang time when compared with the control group, indicating a decline in muscle strength (Fig. 2).

[7] 88w Histological examination of the cerebellum of control rats showed normally distributed neuronal to glial cells population and no abnormality was observed. Acyclovir treatment (10 and 20 mg/kg) resulted in disruption of the neuronal architecture as neuronal damage and loss were observed. The neuropil showed aberrations with increased staining intensity and the presence of reactive gliosis. Cerebellum of acyclovir-treated rats (40 mg/kg) showed astrocyte reactive changes and severe gliosis. The astrocytes appear hypertrophied with multiple branching processes, giving them a starlike appearance. Also, control rats had cortical tissue with

[8] 90w In the cerebellum, IL-6 and TNF-α levels were significantly increased while IL-10 was significantly lowered by acyclovir (20 and 40 mg/kg). However, the cerebellar IL-1β levels were not affected by all doses. In the prefrontal cortex, acyclovir had similar effects as it had on the cerebellum; IL-1β was not significantly altered but pro-inflammatory cytokines IL-6 and TNF-α concentrations were significantly increased while anti-inflammatory cytokine IL-10 level was significantly lowered by acyclovir. In the basal ganglia, acyclovir (40 mg/kg) significantly increased IL-6 and TNF-α levels while IL-10 was decreased (Fig. 4).