IGF-1/GLP-1 Signaling Activator 4-hydroxyisoleucine (4-HI) Prevent Neurobehavioral and Neurochemical Defects in Methylmercury-induced Experimental Model of ALS: Insights From CSF, Blood Plasma and Brain Homogenate Samples in Rats


 Amyotrophic lateral sclerosis (ALS) is the most prevalent adult motor neuron disease, characterized by progressive neuromuscular atrophy, heterogeneous muscle wasting, weakness, and behavioural despair-like symptoms, which are frequently associated with cognitive impairments. The neuropathological hallmarks of MeHg-induced ALS include oligodendrocyte destruction, myelin basic protein (MBP) depletion, and white matter degeneration, which ultimately leads to demyelination and motor neuron death. Numerous research studies have demonstrated that IGF-1/GLP-1 signaling dysregulation plays a significant role in ALS progression as it triggers programmed neuronal cell death, myelin sheath destruction, demyelination, glutamate excitotoxicity, pro-inflammatory cytokine release, and neuroinflammation. 4-hydroxyisoleucine (4-HI) is a unique bioactive amino acid derived from Trigonella foenum graecum, with insulin-mimetic and insulin-sensitizing properties in animal models. The objective of this study was to explore the neuroprotective potential of 4-HI on behavioural, molecular, neurochemical, and gross pathological alterations in MeHg-treated ALS-like rats, with a particular focus on its influence on IGF-1/GLP-1 upregulation in the brain. Furthermore, we investigated the effect of 4-HI on the concentration of myelin basic protein (MBP) in rat brain homogenate and CSF, and specific cell death markers such as caspase-3, Bax, and Bcl-2 in rat brain homogenate and blood plasma samples. In order to investigate the neurobehavioral abnormalities, rats were evaluated for muscular strength via the grip strength test (GST), locomotor deficits using open-field task (OFT), depressive behaviour with forced swim test (FST), and spatial learning in the Morris water maze (MWM) task. Chronic oral treatment with 4-HI at doses of 50 mg/kg and 100 mg/kg was given from days 22nd to 42nd of the experimental protocol to alleviate ALS-like symptoms in the MeHg model of rats. In addition to cellular, molecular, apoptotic, and neuroinflammatory assessments, neurotransmitter levels and oxidative stress indicators were examined in rat brain homogenates. The results of this study consistently show that 4-HI increases the level of neurotrophic growth factors such as IGF-1 and GLP-1 restores the altered neurochemical levels, and potentially prevents ALS-like gross pathological anomalies, including demyelination volume in the rat brain in a dose-dependent manner.

authors concluded that this neuroendocranial action of 4-HI possibly involved multiple stress relieving mechanisms .
Insulin resistance in the brain is associated with blunted responsiveness of IGF-1/GLP-1 signaling pathways, characterized by decreased insulin-stimulated glucose uptake due to altered translocation of glucose transporter-4 (GLUT-4) from the intracellular compartment to the plasma membrane. In a study, a positive modulator of GLP-1 signaling, named N55, was puri ed and isolated from fenugreek seeds. It was reported that N55 promoted GLP-1-dependent cAMP production and GLP-1R endocytosis in a dosedependent and saturable manner. N55 bound to GLP-1R and played a role in facilitating glucose excursion in diabetes and reducing glycated hemoglobin levels in humans. It was also found that N55 speci cally enhanced GLP-1 potency by more than 40-folds to stimulate cAMP production. (King et al., 2015).
Furthermore, IGF-1 receptors need PI3K-Akt dependent pathways to exert their anti-apoptotic and nerve growth-stimulating effects. 4-HI increases tyrosine phosphorylation of both insulin receptor-β and insulin receptor substrate-1. 4-HI also activates AMP-activated protein kinase and insulin-dependent PI3-kinase pathway, which is necessary for neuroprotective functions of nerve growth factor (NGF) and brain-derived neurotrophic factors (BDNF). As a result, the interactions between NGF, 4-HI, and insulin may provide a credible explanation for its neuroprotective properties (Gautam et al., 2016).
In the light of the above ndings, we hypothesize that 4-HI can upregulate the impaired IGF-1/GLP-1 target signaling mechanisms and hence, can ameliorate the neurodegenerative alterations in MeHginduced ALS-like rats via its potential target-modulating properties. In addition, 4-HI could be associated with other standard drug treatments as a novel therapeutic intervention for increasing the life expectancy of patients with demyelinating disorders such as ALS. Therefore, these promising pharmacological effects provoked us to investigate and evaluate the neuroprotective pro le of 4-HI in rat brain homogenate, blood plasma, and CSF samples, with an objective that these biological samples can be utilized as effective and reproducible diagnostic biomarkers during the early stages of neural degeneration.

Experimental animals
The Central Animal House, ISF College of Pharmacy, Moga, Punjab, provided 39 adult Wistar rats (250-300 grams, aged six months, total animals = 36, total experiment groups = 06, each group comprising six rats of either sex). The animals were kept in polyacrylic cages (38cm×32cm×16cm; 3-4 rats per cage) with a wire mesh top and soft paddy husk bedding. They were acclimatized to standard husbandry conditions of 12 hours light/dark constant cycle with normal chow diet as dried pellets and ltered water ad libitum, maintained at a controlled temperature of 23 ± 2℃ and relative humidity 65-70%. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) with registration no. 816/PO/ReBiBt/S/04/CPCSEA and protocol no. ISFCP/IAEC/CPCSEA/Meeting No.27/2020/Protocol No.455 in compliance with the guidelines of the Government of India. Animals were adapted for a week to standard laboratory conditions before the commencement of the experiments.

Drugs and chemicals
MeHg was purchased from Sigma-Aldrich (USA). The protocol drug, 4-HI (amorphous, hydrophilic powder), was acquired as ex-gratia sample from BAPEX Pharmaceuticals, India. All of the other compounds employed in the study were of analytical grade. An aqueous solution of 4-HI with 2% ethanol was orally administered in the volume of 10 ml/kg (Siddiqui et al., 2021;Gaur et al., 2012).

Protocol schedule of animal experimentation
The total duration of the experimental protocol schedule was 42 days. From day 1st to 21st, MeHg was regularly administered via oral route at the dose of 5 mg/kg. The protocol drug, 4-HI, was administered at the dose of 50 mg/kg and 100 mg/kg from day 22nd until the 42nd day of the study duration. Animals were randomly assigned into six groups (n = 6 animals per group). Group 1: Normal control, Group 2: Vehicle control, Group 3: (4-HI100 mg/kg, perse, p.o.), Group 4: (MeHg5 mg/kg, p.o.), Group 5: (MeHg5 mg/kg, p.o. + 4-HI50 mg/kg, p.o.), and Group 6: (MeHg5 mg/kg, p.o. + 4-HI100 mg/kg, p.o.). Various neurobehavioral assessments like grip strength test, open eld test, forced swim test, and Morris water maze task was carried out between days 1st to 42nd of the study duration. At the end of the protocol schedule, i.e. (day 43rd ), animals were deeply anesthetized with sodium pentobarbital (270 mg/ml, i.p.), 2.5 ml of blood was collected from the rats through the retro-bulbar puncture, and 100µl of CSF was collected from the cistern magna and stored at -80ºC for future use. Animals were then sacri ced by decapitation, and their whole brains were carefully removed from the skull for various neurochemical estimations and gross morphological analysis. Fresh whole brains isolated from the rats were weighed, further homogenized in phosphate buffer saline (PBS). All the biochemical estimations were performed in brain homogenate, CSF, and blood plasma samples. Figure 1 shows a schematic representation of the protocol paradigm and time course of experimentation.

Experimental animal model of methylmercury-induced ALS-like rats
The MeHg-induced experimental model of ALS in rats was established according to the method validated by (Alam et al., 2020, Minj et al., 2021. Experimental rats were administered with MeHg orally at 5 mg/kg for three consecutive weeks. Pre-clinically, MeHg causes neurological impairment similar to an experimental animal model of ALS and is considered a valid model for studying the pathophysiological changes similar to ALS. The selection of MeHg as a possible neurotoxicant is also based on the evidence that it has been related to producing ALS-like signs. It produces glutamate-mediated excitotoxicity, calcium-dependent neurotoxicity and induces ALS-like phenotype (Johnson et al., 2011). Thus, in this study, MeHg was used as a neurotoxin to observe behavioral impairments, neurochemical imbalances, and gross morphological alterations in ALS-like rats.
Assessment of relative brain-body weight ratio On the 42nd day of the experimental schedule, the brain weights of all rats were recorded after their sacri cation. A ratio of the brain to nal body weight was calculated to examine the alterations in absolute brain mass of MeHg treated ALS-like rats with respect to their body weight. This assessment can detect the chronic, neurotoxic effects of MeHg exposure on the brain and hence, can potentially establish a link between MeHg exposure and loss of brain-body weight. The relative brain-body weight ratio was calculated using the formula; (brain weight/body weight) ×100 (Aminu et al., 2019).

Grip strength test (GST)
A grip strength test was used to evaluate the forelimb and hindlimb strength in different treatment groups. The test was performed on days 1st, 22nd, 33 rd, and 42nd of the protocol. The rats were allowed to grasp the metal bar/grid with their forepaws and then pulled backward by the tail in the horizontal plane. The force applied to the grid before the rat lost grip was recorded as the maximal peak force. This force was measured in kgf (Chatillon, USA) (Mehan et al., 2020).

Open eld test (OFT) assessment
Locomotor activity and anxiety-like behaviour in the rats were assessed with the open-eld task. OFT assessment is a common measure of exploratory behavior and general locomotor activity in rodents. Experimental animals were tested for their locomotor activity and number of rearings on 1st, 11th, 21st, 31 st, and 41st treatment days. Each animal was placed at the center of the enclosure (70 cm long× 70 cm wide× 60 cm high), and the number of boxes/segments crossed by the animal with their four paws in ve minutes was recorded as a measure of their locomotor activity. The number of rearing outcomes indicated by the animal throughout the ve-minute sessions was evaluated as an indicator of emotionality (degree of anxiety). (Alam et al., 2020) Forced swim test (FST) FST is a behavioral despair test used to evaluate depressive-like behavior in animals. It measures an animal's susceptibility to the acute stress of water by assessing their response to the threat of drowning.
Each animal was tested for a forced swim on days 21st, 28th, 35 th, and 42nd of the study by placing them individually in a clear, plastic cylindrical tank lled up to 15 cm with tap water (at 23-25°C temperature; height: 40cm; diameter: 18cm). The immobility time and swimming time (movement usually horizontal throughout the swim chamber) were manually scored in each trial during the total period of ve minutes. The rats were scored for immobility duration when they ceased struggling and remained oating motionless in the upright position by making only small movements to keep their head above water. Immediately following the test, the rats were wrapped in a dry towel and placed under a heating lamp to dry (Sharma et al., 2019).

Morris water maze (MWM) task
The MWM test is a robust and reliable test strongly correlated with hippocampal synaptic plasticity and cognitive dysfunctions. This task was performed to evaluate spatial learning and memory in rats (Kaur et al., 2015). The working memory of the rats was analyzed by evaluating their escape latency time (ELT) on the 39 th, 40 th, and 41st days of the protocol schedule. Rats were allowed to navigate freely around the perimeter of an open swimming arena to locate a submerged escape platform. Twenty-four hours after the acquisition phase, the time taken by the rats to nd a platform and escape the maze was recorded as ELT (long-term memory).
On the 42nd day, a probe/memory test was conducted by removing the platform. The rats were allowed to swim freely in the pool of water to nd the platform (which was previously present) within 120 seconds, and the time spent by the animal in the target quadrant (TSTQ) zone to locate the platform was recorded by a video system. The TSTQ indicated the degree of relative memory consolidation (reference memory) in rats that had occurred following spatial learning (Duggal et al., 2020).

Neurochemical parameters
Collection and preparation of biological samples On day 43rd of the experiment, 2.5 ml of blood was collected from anesthetized rats through retro-bulbar puncture from the orbital venous plexus by inserting a capillary tube medially into the rat eye. Blood from the plexus was collected into a sterile Eppendorf tube via the capillary action through gentle rotation and retraction of the tube (Boynton et al., 2020). The blood samples were then centrifuged at 10,000×g for 15 minutes to separate the plasma, and the supernatant was carefully stored in a deep freeze (at -80 o C) for further use.
Following blood collection, rats were deeply anesthetized with sodium pentobarbital (270mg/ml, i.p.) and subjected to caudal incision, translucent duramater was exposed, and a 30 gauge needle was gently placed at 30 angle into the cisterna magna (Pegg et al., 2010). Approximately 100µL CSF was carefully ejected into 0.5ml sterile Eppendorf tube using the suction pressure of a 1ml tuberculin syringe attached to a needle, and the collected sample was frozen at 80 C until analyzed by ELISA (Rubio et al., 2011).
Immediately after CSF collection, rats were sacri ced by decapitation; whole brains were isolated from the skull with the utmost care, freshly weighed and washed with ice-cold, isotonic saline solution, and then homogenized with 0.1M (w/v) of chilled PBS (pH = 7.4). The rat brain homogenate was then centrifuged at 10,000×g for 15 minutes, the supernatant was separated, and the aliquots were preserved. The samples were deep-freezed at -80ºC to be used as and when required for various biochemical estimations.

Assessment of cellular and molecular markers
The concentration of IGF-1 was determined in rat brain homogenate (Ola et al., 2014) and CSF (Salehi et al., 2008) using ELISA commercial kits (Elabsciences, China) as per the manufacturer's instructions. Similarly, GLP-1 levels were also assessed in brain homogenate (Candeias et al., 2018) and CSF obtained from rats. Furthermore, the level of myelin basic protein (MBP) was measured in rat brain homogenate (Minj et al., 2021) and CSF samples (Ohta et al., 2002). The levels of IGF-1 and GLP-1 proteins in brain homogenate and CSF were represented as pg/mg protein and ng/ml protein, respectively. For MBP, the values were expressed as µg/mg protein in brain homogenate and ng/L protein in CSF.

Assessment of apoptotic markers
The level of pro-apoptotic markers like caspase-3, Bax, and anti-apoptotic Bcl-2 was assessed using ELISA assay kits (Elabsciences, China). Quanti cation tests for caspase-3 were performed in rat brain homogenate (Rahi et al., 2021) and blood plasma (Song et al., 2015) as per the given standard procedures. Similarly, the level of Bax was measured in brain homogenate (Tiwari et al., 2021) and blood plasma samples (Budworth et al., 2012). Moreover, the concentration of Bcl-2 protein was also evaluated in rat brain homogenate  and blood plasma (Tao et al., 2016) obtained from rats. The caspase-3 level was measured in brain homogenate as nM/mg protein and blood plasma as ng/ml. The level of Bax/Bcl-2 in brain homogenate and blood plasma were represented in ng/mg and ng/ml, respectively.

Assessment of neurotransmitter levels
Glutamate is the major excitatory amino acid neurotransmitter in the CNS, which plays a key role in brain development, learning and memory. At rst, the derivatization of ortho-phthalaldehyde/βmercaptoethanol was done, followed by the quanti cation of GABA and glutamate levels. The method of Alam and co-workers has been followed for this quantitative analysis in the brain tissue samples. The amount of GABA/glutamate present in the brain homogenate supernatant was expressed as ng/mg protein (Alam et al., 2020).
A diagnostic kit (KRISHGEN Diagnostics, India) was used to measure acetylcholine (Ach) levels. All the reagents and samples were freshly prepared as described in the kit. The optical density of the reaction mixture was determined at 540nm with the help of a microtiter plate. The level of Ach present in the brain homogenate was expressed as ng/mg protein .
The serotonin (5-HT) level was estimated with the help of HPLC using a C18 reversed-phase column and an electrochemical detector (ECD) at + 0.75 V, with sensitivity ranging from 5 to 50 nA. The mobile phase consisted of sodium citrate buffer (pH = 4.5) and acetonitrile in the ratio 87:13, v/v. The concentration of serotonin was estimated using a standard with a concentration of 10-100 mg/ml from the standard curve. The level of the 5-HT present in the rat brain homogenate was expressed as ng/mg protein. (Tiwari et al., 2021).

Assessment of in ammatory cytokine levels
The level of TNF-α was quanti ed in brain homogenate (Khera et al., 2019) and blood plasma (Wu et al., 2016) by using rat TNF-α immunoassay kits (KRISHGEN BioSystem, USA). Similarly, the concentration of IL-1β was also determined in rat brain homogenate (Chen et al., 2016) and blood plasma samples (Speaker et al., 2014) by using ELISA commercial kits. These in ammatory cytokine levels in rat brain homogenate and blood plasma were expressed as pg/mg protein and pg/ml protein, respectively.

Assessment of oxidative stress markers
The AchE level in the brain homogenate sample was determined spectrophotometrically. The assay mixture contained brain homogenate supernatant, 0.01M sodium phosphate buffer (pH = 8), acetylthiocholine iodide, and DTNB (Ellman's reagent). The enzymatic activity of supernatant was expressed as µM/mg protein (Bala et al., 2015).
To measure the level of LDH in the rat brain homogenate, a diagnostic kit (Coral diagnostics, India) was used, and the amount of LDH was quanti ed as Units/L (Mehan et al., 2017).
The amount of MDA in the homogenate was determined using the spectrometric method at 532nm, after its reaction with the thiobarbituric acid (TBA) reagent. The TBARS concentration was calculated using TEP as a standard. The MDA concentration was expressed as nM/mg protein .
The nitrite (NO 2 ) level was determined by a colorimetric assay using Greiss reagent, and the absorbance was determined at 540nm with a UV spectrometer. The nitrite concentration in the supernatant was determined via a sodium nitrite standard curve and expressed as µM/mg protein (Mehan et al., 2011).
The amount of antioxidant SOD was quanti ed based on the ability of SOD to inhibit the self-oxidation of epinephrine in alkaline conditions. A colored adrenochrome is formed during this reaction, while superoxide anion-radicals are formed as intermediate products of this process, and the absorbance was measured with a spectrometer at 480nm. The level of SOD in the sample was expressed as µM/mg protein (Singh et al., 2018).
The antioxidant GSH content in brain homogenate was determined based on the concept of DTNB reduction by thiol compounds (mainly GSH) to the colored 2-nitro-5-mercaptobenzoic acid. The yellow color that developed was measured immediately at 412nm using a UV spectrometer. The concentration of GSH in the supernatant was expressed as µM/mg protein (Deshmukh et al., 2009). Assessment of gross pathology and demyelination volume in rat brain Animals were decapitated and sacri ced on day 43rd after the completion of the experimental schedule.
Fresh, whole brains were isolated from the skull, freshly weighed, and immediately preserved in ice-cold saline for gross pathological analysis. Coronal sections of the brain were taken after macroscopic examination of the whole rat brain (Kumar et al., 2021). Cerebral hemispheres were sectioned into 2mm thick parallel coronal slices and placed on glass slides for morphological and pathological assessment. A digital camera was used for macroscopic inspection of the entire striatum. . The volume of the demyelinated region (mm 3 ) for each brain segment was estimated from the greyish region around the striatum. In each coronal segment, the extent of white matter degeneration was determined by calculating the demyelination volume (length×breadth×height) (Minj et al., 2021).

Statistical analysis
Data generated were analyzed using two-way analysis of variance (ANOVA) followed by post-hoc Bonferroni's test, and one-way ANOVA repeated measures followed by post-hoc Tukey's multi comparison test to examine the differences between various treatment groups. Two-way ANOVA was used to analyze the body weight and all the behavioral parameters. In contrast, one-way ANOVA was used to analyze the relative brain-body weight ratio, demyelination volume, biochemical parameters, and TSTQ analysis. P < 0.001 was considered statistically signi cant. Data was found to be normal, and the sample size was calculated by checking the normality distribution via the Kolmogorov Smirnov test. The statistical analysis was done using GraphPad Prism software version 5.03 for Windows (GraphPad Software, San Diego, CA, USA). All the statistical results are presented as the mean and standard error of mean (SEM).

Results
Effect of 4-hydroxyisoleucine in the restoration of weight variations in methylmercury-induced ALS-like rats Improved body weight after long term administration of 4-hydroxyisoleucine To determine the role of 4-HI in MeHg-treated ALS-like rats, alterations in body weight were assessed on days 1st, 7th, 14th, 21st, 28th, 35 th, and 42nd of the experimental protocol schedule. On days 1st and 7th, there was no signi cant difference between all the groups. On days 14th, 21st, 28th, 35 th, and 42nd, a gradual decline in bodyweight was found in rats treated with MeHg compared to normal control, vehicle control, and 4-HI100 perse treated groups. Continuous oral administration of 4-HI50 mg/kg and 100 mg/kg signi cantly and dose-dependently improved the bodyweight when compared with MeHg-treated ALS-like rats on 14th, 21st, 28th, 35 th, and 42nd days of the experimental schedule [Two-way ANOVA: F(30,180) = 187.8, P < 0.001]. On days 35th and 42nd, 4-HI100 mg/kg treated ALS-like rats showed signi cant improvements in body weight restoration as compared to the 4-HI50 mg/kg treatment rats ( Fig. 2). Improvement in relative brain-body weight ratio after long term administration of 4-hydroxyisoleucine On the 42nd day of the experimental procedure, the relative brain-body ratio was analyzed to investigate the neuroprotective effect of 4-HI in MeHg-treated ALS-like rats. The normal and vehicle control groups showed no signi cant difference in the relative brain-body weight ratio compared to 4-HI perse treated rats. Moreover, when compared to the normal control, vehicle control, and 4-HI100 perse groups, MeHginduced ALS-like rats demonstrated a steady decline in relative brain-body weight ratio. Long-term oral administration of 4-HI at the doses of 50 mg/kg and 100 mg/kg substantially enhanced the relative brainbody weight ratio compared to the MeHg-treated rats [One-way ANOVA: F(5,25) = 0.632, P < 0.001]. In comparison to the 4-HI 50 mg/kg treated group, 4-HI100 mg/kg demonstrated a considerable increase in relative brain-body weight ratio. These results re ect that 4-HI can potentially restore the absolute brain mass alterations with respect to the body weight (Fig. 3).
Effect of 4-hydroxyisoleucine in the amelioration of neurobehavioral abnormalities in methylmercuryinduced ALS-like rats Improved grip strength after long term administration of 4-hydroxyisoleucine Grip strength tests were performed on days 1st, 22nd, 32nd, and 42nd of the protocol schedule to assess the potential in uence of 4-HI on muscle strength. On day 1st, a non-signi cant difference was observed between all the treatment groups. The oral treatment with MeHg for 21 days consecutively led to a gradual decrease in grip strength force on days 22nd, 32 nd, and 42nd as compared to normal control, vehicle control, and 4-HI100 perse groups. Prolonged administration of 4-HI 50 mg/kg and 100 mg/kg showed a remarkable improvement in the grip strength force on days 22nd, 32 nd, and 42nd when compared to MeHg-treated ALS-like rats [Two-way ANOVA: F(15,90) = 119.6, P < 0.001] Among these, 4-HI100 mg/kg was more effective than 4-HI 50 mg/kg in the restoration of the grip strength on days 32nd and 42nd (Fig. 4).

Improved locomotion and restored anxiety-like behavior after long term administration of 4hydroxyisoleucine
In order to evaluate the general locomotor activity and examine the level of anxiety in rats, assessments of the number of boxes crossed and evaluation of the number of rearings were performed on days 1st, 11th, 21st, 31 st, and 41st of the protocol duration. On the rst day, there was no signi cant difference between the groups. The MeHg-treated ALS-like rats showed a progressive decline in the number of boxes crossed on days 11th, 21st, 31 st, and 41st and a considerable decrease in the number of rearings in comparison with normal control, vehicle control, and 4-HI100 perse groups. Consequently, the regular administration of 4-HI 50 mg/kg and 100 mg/kg signi cantly enhanced the locomotor activity [Two-way ANOVA: F(20,120) = 35.71, P < 0.001] and restored the level of anxiety (rearing score) [Two-way ANOVA: F(20,120) = 16.16, P < 0.001] as compared to MeHg-treated ALS-like rats. On days 31st and 41st, it was observed that 4-HI100 mg/kg considerably enhanced locomotion and improved the rearing scores when compared to the 4-HI 50 mg/kg treatment group (Fig. 5, Fig. 6).
Decreased depression-like behavior after long term administration of 4-hydroxyisoleucine The forced swim test (FST) was performed on days 21st, 28th, 35th, and 42nd of the protocol sequence to explore the despair behavior in ALS-like rats. The meHg-treated group showed depressive-like behavior and a gradual increase in immobility time when compared to normal control, vehicle control, and 4-HI100 perse treated rats. Long-term oral administration of 4-HI50 mg/kg and 4-HI100 mg/kg led to a considerable decline in the immobility time on days 28th, 35th, and 42nd when compared to the MeHgtreated group [Two-way ANOVA: F(15,90) = 87.32, P < 0.001] Among them, 4-HI at a dose of 100 mg/kg was found to be more effective than 4-HI 50 mg/kg in reducing immobility time in rats on days 28th, 35th, and 42nd (Fig. 7).
Improved memory and cognition after long term administration of 4-hydroxyisoleucine Escape latency time (ELT) was evaluated on the 39th, 40th, and 41st days of the treatment schedule. MeHg treated ALS-like rats showed a gradual increase in ELT compared to the normal control, vehicle control, and 4-HI100 perse groups. Chronic treatment with 4-HI 50 mg/kg and 100 mg/kg resulted in a signi cant decrease in ELT with respect to the MeHg-treated ALS-like group [Two-way ANOVA: F(10,60) = 1.891, P < 0.001]. 4-HI100 mg/kg led to a marked decline in ELT compared to 4-HI 50 mg/kg treatment, which suggests that 4-HI at a dose of 100 mg/kg is more e cient in improving memory acquisition ALSlike rats (Fig. 8).
TSTQ was also examined at the end of the treatment schedule (on day 42nd ) to determine the degree of memory consolidation or retention of memory in rats that had occurred following learning. MeHg-induced ALS-like rats demonstrated a substantial decrease in time spent in the target zone than the normal control, vehicle control, and 4-HI100 perse groups. Long-term oral treatment with 4-HI 50 mg/kg and 100 mg/kg comparatively increased the TSTQ with respect to MeHg treated ALS-like group [One-way ANOVA: F(5,25) = 1.967, P < 0.001]. Relatively, 4-HI100 mg/kg showed a signi cant rise in TSTQ compared to 4-HI 50 mg/kg treated ALS-like rats (Fig. 9).

Effect of 4-hydroxyisoleucine on neurochemical alterations in methylmercury-induced ALS-like rats
Increased level of IGF-1 after long-term administration of 4-hydroxyisoleucine IGF-1 levels were assessed in rat brain homogenate and CSF at the end of the experimental protocol. Oral administration of MeHg resulted in a signi cant decrease in the level of IGF-1 as compared to normal control, vehicle control, and 4-HI100 perse groups. Chronic oral administration of 4-HI at the doses of 50 mg/kg and 100 mg/kg remarkably increased the IGF-1 level in brain homogenate [One-way ANOVA: F(5, 25) = 5.595, P < 0.001] and CSF samples [One-way ANOVA: F(5,25) = 0.971, P < 0.001]. Moreover, 4-HI100 mg/kg was more e cient than the 4-HI 50 mg/kg treated group in restoring the IGF-1 protein level in both rat brain homogenate and CSF samples.
Increased level of GLP-1 after long-term administration of 4-hydroxyisoleucine GLP-1 levels in rat brain homogenate and CSF were measured at the end of the treatment schedule to explore whether 4-HI is involved in activating the GLP-1 signaling. MeHg-induced ALS-like rats illustrated a remarkable decrease in the level of GLP-1 as compared to normal control, vehicle control, and 4-HI100 perse treated groups. Prolonged 4-HI50 mg/kg and 100 mg/kg treatment led to a signi cant and dosedependent increase in the GLP-1 concentration in brain homogenate [One-way ANOVA: F (5,25) = 2.009, P < 0.001] and CSF [One-way ANOVA: F(5,25) = 2.021, P < 0.001] respectively. Moreover, the 4-HI100 mg/kg dose was more e cient in restoring the level of GLP-1 in comparison with 4-HI50 mg/kg. Restored level of myelin basic protein after long-term administration of 4-hydroxysoleucine The level of myelin basic protein (MBP) was assessed in rat brain homogenate and CSF using a commercial ELISA kit at the end of the protocol schedule. MBP levels were shown to be considerably lower in brain homogenate and higher in CSF samples in MeHg toxin-treated ALS-like rats compared to the normal control, vehicle control, and 4-HI100 perse groups. In comparison to the MeHg-treated group, long-term treatment with 4-HI at doses of 50 mg/kg and 100 mg/kg enhanced MBP level in rat brain homogenate [One-way ANOVA: F(5,25) = 0.7205, P < 0.001] but lowered the MBP level in CSF [One-way ANOVA: F(5,25) = 0.5460, P < 0.001]. It was also found that 4-HI100 mg/kg administration effectively restored the altered MBP levels than 4-HI50 mg/kg administration, implying that 4-HI is bene cial in restoring myelin degradation and lowering demyelination in a dose-dependent manner (Table 1). Decreased level of caspase-3, Bax, and increased Bcl-2 levels after long-term administration of 4hydroxyisoleucine The neuronal cell death markers like caspase-3, Bax, and Bcl-2 were evaluated in rat brain homogenate and blood plasma at the end of the protocol schedule. Long-term oral MeHg administration resulted in a considerable rise in pro-apoptotic markers like caspase-3 and Bax in rat brain homogenate and blood plasma. In contrast, oral treatment with MeHg for 21 days led to a notable decline in anti-apoptotic Bcl-2 protein level in rat brain homogenate and blood plasma compared to normal control, vehicle control, and 4-HI perse treated groups. Chronic oral administration of 4-HI at doses of 50 mg/kg and 100 mg/kg substantially reduced the level of caspase-3 in brain homogenate [One-way ANOVA: F(5,25) = 0.9588, P < 0.001] and blood plasma [One-way ANOVA: F(5,25) = 0.5116, P < 0.001] respectively.
Likewise, continuous oral treatment with 4-HI at doses of 50 mg/kg and 100 mg/kg remarkably decreased the level of pro-apoptotic marker Bax in rat brain homogenate [One-way ANOVA: F(5,25) = 3.902, P < 0.001] and blood plasma [One-way ANOVA: F(5,25) = 1.642, P < 0.001]. However, regular oral administration of 4-HI at doses of 50 mg/kg and 100 mg/kg for 21 days consecutively led to a considerable increase in the level of Bcl-2 protein in brain homogenate [One-way ANOVA: F(5,25) = 7.161, P < 0.001] and blood plasma [One-way ANOVA: F(5,25) = 0.9438, P < 0.001] concerning the MeHg treated ALS-like rats. Furthermore, 4-HI100 mg/kg treatment was more effective than 4-HI50 mg/kg treatment in restoring the abnormal levels of apoptotic markers in ALS-like rats (Table 2). Restoration of neurotransmitter levels after long-term administration of 4-hydroxyisoleucine Amount of neurotransmitters such as GABA, glutamate, acetylcholine, and serotonin in the rat brain homogenate were estimated at the end of the protocol schedule. Oral administration of toxin MeHg for 21 days consecutively resulted in a substantial decrease in GABA, acetylcholine, and serotonin levels. In contrast, continuous oral MeHg intoxication led to a signi cant increase in the glutamate concentration in brain homogenate compared with normal control, vehicle control, and 4-HI100 perse treated rats.
However, oral administration of 4-HI at doses of 50 mg/kg and 100 mg/kg lowered the concentration of glutamate [One-way ANOVA: F(5,25) = 1.372, P < 0.001] in rat brain homogenate in contrast to the MeHg treated ALS-like group. Among these, the most signi cant improvements were observed in 4-HI100 mg/kg treated ALS-like rats versus the 4-HI50 mg/kg treated rats in the restoration of neurotransmitter imbalance (Table 3).

Reduction in neuroin ammatory cytokines after long-term administration of 4-hydroxyisoleucine
To examine the neuroprotective effect of 4-HI on the pro-in ammatory cytokines, we evaluated the level of TNF-α and IL-1β in the whole brain homogenate and blood plasma of rats. Treatment with 4-HI at doses of 50 mg/kg and 100 mg/kg signi cantly lessened the TNF-α level in rat brain homogenate [Oneway ANOVA: F(5,25) = 3.181, P < 0.001] and blood plasma [One-way ANOVA: F(5,25) = 1.682, P < 0.001] respectively. Similarly, chronic oral treatment with 4-HI50 mg/kg and 4-HI100 mg/kg remarkably lowered the level of IL-1β in brain homogenate [One-way ANOVA: F(5,25) = 2.058, P < 0.001] and blood plasma [One-way ANOVA: F(5,25) = 1.370, P < 0.001] as opposed to the MeHg toxin administered ALS-like rats. Meanwhile, 4-HI100 mg/kg showed a marked improvement in reducing the level of these neuroin ammatory cytokines as compared to 4-HI50 mg/kg dose (Table 4). In comparison to 4-HI50 mg/kg, 4-HI100 mg/kg treatment signi cantly reduced the level of oxidative stress markers while also restoring antioxidant level in a dose-dependent manner (Table 5). Effect of 4-HI on gross pathological alterations and demyelination volume in MeHg-treated ALS-like rats Improvement in whole-brain morphological alterations after long-term administration of 4hydroxyisoleucine The normal control, vehicle control, and 4-HI perse treated groups depicted appropriate brain morphology, adequate shape, and unaltered size. MeHg-treated rat brains showed shrinkage in size and reduced overall brain mass compared to normal control, vehicle control, and 4-HI perse treated groups. Continuous oral administration of 4-HI at doses of 50 mg/kg and 100 mg/kg was observed to be e cient in restoring the alterations in brain weight and effective in reducing cortical degeneration. However, 4-HI100 mg/kg treated ALS-like rats showed signi cant improvements in whole-brain morphology compared to the 4-HI50 mg/kg treatment rats (Fig. 10).
Reduced pathological abnormalities in brain sections after long-term administration of 4hydroxyisoleucine The normal control, vehicle control, and 4-HI100 perse treated rat brain sections showed distinct appearance with clearly de ned basal ganglia, cerebral cortex, and hippocampal tissues. However, the brain sections of rats treated with MeHg demonstrated a considerable reduction in several cortical tissues, myelin degradation, white matter degeneration, and demyelination as compared to normal control, vehicle control, and 4-HI100 perse treated rats. Persistent treatment with 4-HI50 mg/kg and 4-HI100 mg/kg remarkably reversed these morphological alterations in brain sections and restored demyelination, measured by demyelination volume and MBP level assessment. Among these, 4-HI100 mg/kg was more effective than 4-HI50 mg/kg in amelioration of the pathological abnormalities in brain sections (Fig. 11).
Reduced demyelination volume after long-term administration of 4-hydroxyisoleucine Long-term oral administration of neurotoxin MeHg for 21 days substantially induced the destruction of the myelin content instead of normal control, vehicle control, and 4-HI100 perse treated animals. Treatment with 4-HI at doses of 50 mg/kg and 100 mg/kg considerably reduced demyelination volume relative to MeHg-treated ALS-like rats [One-way ANOVA: F(5,25) = 1.627, P < 0.001] Consequently, 4-HI100 mg/kg depicted a noticeable, dose-dependent improvement in myelin restoration as compared to 4-HI50 mg/kg treated ALS-like rats, which was further evaluated by demyelination volume (Fig. 12).

Discussion
In the present research, the MeHg intoxication-induced experimental rat model was used to study the pathological alterations similar to ALS. Evidence from different animal studies suggests that MeHg exposure may contribute to ALS progression (Minj et al., 2021, Alam et al., 2020, Praline et al., 2007. MeHg exposure in a mouse model with mutant human SOD1 gene over expression resulted in ALS-like symptoms such as the early onset of hind limb impairment (Johnson and Atchison, 2008).
MeHg being lipophilic, readily diffuses from the blood into the CNS, across the blood-brain barrier (BBB) as a cysteine complex via the L-type neutral amino acid carrier transport (LAT) system (Khan et al., 2011).
It combines covalently with the sulfhydryl (thiol) groups in the plasma cholinesterase, leading to enzyme inhibition (Farina et al., 2011). The mercury atom in MeHg interacts directly with the thiol group of reduced glutathione (GSH), leading to the production of the GS-HgCH 3 complex, which is quickly eliminated from the body, resulting in antioxidant depletion (Stringari et al., 2008). MeHg causes glutamate-mediated excitotoxicity by overstimulating NMDA receptors and decreasing the expression of excitatory amino acid transporters (EAATs) in astrocytic synaptic clefts. (Johnson et al., 2011). It also triggers calcium-dependent neurotoxicity via intracellular Ca 2+ dyshomeostasis, leading to further generation of oxidative stress (Ceccatelli et al., 2010), and eventually, motor neuron death (Nascimento et al., 2008). MeHg disrupts the mitochondrial electron transport chain (ETC), induces cytochrome-c release, leading to increased ROS-induced neurotoxicity (Mori et al., 2011).
In this study, we investigated that 4-HI exerts neuroprotective effects against MeHg treated ALS-like rats by conducting various neurobehavioral tests. Furthermore, we also evaluated the levels of cellular and molecular markers, apoptotic markers, neurotransmitters, pro-in ammatory cytokine levels, and oxidative stress indicators in rat brain homogenate, blood plasma, and CSF.
Insights from our behavioral and biochemical results suggest that long-term, oral administration of 4-HI at the doses of 50 mg/kg and 100 mg/kg elicited a bene cial neuroprotective effect against motor dysfunctions and neurological de cits in the MeHg model of ALS-like rats.
Bodyweight analysis and several neurobehavioral evaluations were performed to investigate the neuroprotective effect of 4-HI on neuromotor dysfunctions caused by continuous MeHg administration.
Our results indicate that long-term exposure to 5 mg/kg MeHg treatment resulted in a marginal reduction in animal body weight as well as a signi cant decrease in the relative brain-body weight ratio. In contrast to the MeHg-treated ALS-like rats, the body weight was gradually restored after continuous oral treatment of 4-HI. A similar trend was observed in the restoration of alterations in the relative brain-body weight ratio. Oral treatment with 4-HI remarkably abolished the substantial declines in a relative brain-body weight ratio in MeHg treated ALS-like rats in a steady, dose-dependent manner.
The grip strength test was used to measure changes in grip strength performance. Our results showed that the muscular grip was signi cantly affected in MeHg treated ALS-like rats. However, in the groups that continuously received 4-HI, the loss in forelimb and hindlimb grip strength signi cantly improved.
The open-eld task (OFT) was used to assess changes in general locomotor activity and exploratory behavior in rats . MeHg exposure resulted in a signi cant reduction in the number of boxes crossed as well as a considerable decrease in the number of rearings. Furthermore, 4-HI therapy remarkably alleviated MeHg-induced locomotive impairment and anxiety-like symptoms in ALS-like rats.
Hence, it is evident from the reduced locomotor activity and signi cant decline in grip strength that MeHg administration may contribute to compromised muscle physiology in ALS-like rats. According to research, rats treated with MeHg exhibit behavioral despair characteristics in forced swim tests (Nabi et al., 2012).
Our ndings suggest that repeated 4-HI treatment eventually abolishes such depressive-like states in MeHg-exposed rats, indicating that it has promising neuroprotective effects on the restoration of brain functioning.
Recent research has also revealed that MeHg has a negative impact on neuronal plasticity, learning, and memory (Santos et al., 2016). In our study, we looked at how 4-HI affected long-term memory and cognitive behavior. MeHg administration considerably increased escape latency time (ELT) and decreased time spent in the target quadrant (TSTQ) in ALS-like rats, demonstrating severe neuromotor de cits and cognitive dysfunctions (Minj et al., 2021). Long-term administration of 4-HI, on the other hand, reversed these neurological abnormalities, indicating marked improvements in memory acquisition and retention.
The effect of 4-HI on IGF-1/GLP-1 signaling was analyzed to determine the cellular and molecular mechanisms associated with the impact of 4-HI in neuroprotection. The IGF-1/GLP-1 signaling pathway regulates neuronal activity, excitotoxicity (Perry et al., 2008), neuroin ammation, oxidative stress (Sukhanov et al., 2007), synthesis of the myelin sheath (Freude et al., 2008), and oligodendrogenesis (Chesik et al., 2008). Previous research has revealed that disruption of the IGF-1 and GLP-1 signaling pathways plays a detrimental role in the progression of ALS pathogenesis and related neurocomplications. The neurodegenerative symptoms of ALS, such as oligodendrocytic degradation, myelin degeneration, glial cell overactivation, immunological dysregulation, and neuronal excitation, are triggered by aberrant IGF-1/GLP-1 signaling .
ELISA was used to determine the levels of cellular and molecular markers such as IGF-1 and GLP-1 in rat brain homogenate and CSF. Oral 4-HI treatment signi cantly restored IGF-1 and GLP-1 levels in rat brain homogenate and CSF. These ndings suggest that 4-HI can potentially modulate the IGF-1/GLP-1 signaling deregulation in damaged neuronal cells (King et al., 2015).
Oligodendrocytes are glial cells that form myelin sheaths in the CNS, which function as electrical insulators and help maintain axonal homeostasis (Marc et al., 2019). Myelination is essentially the multiple wrapping of axons with oligodendrocyte processes induced by a phase change of myelin basic proteins (MBP) from a soluble to a more viscous and cohesive protein meshwork (Aggarwal et al., 2013).
A key pathogenic aspect of ALS etiology is myelin sheath degradation, induced by oligodendrocyte dysfunction and a gradual reduction in MBP levels (Raffaele et al., 2021). Demyelination has been linked to motor dysfunction, neurological de cits, diminished cognitive capacities, and reduced conduction velocity of electrical impulses (Fields., 2008).
Previous research has shown that white matter damage and demyelination can occur prior to motor neuron death and could be used to predict ALS prognosis (Chio et al., 2014). A study looked at oligodendrocyte degeneration in the spinal cords of G93A-SOD1 ALS mice before the disease onset. It was found that, although new oligodendrocytes were produced, they did not mature, resulting in increased demyelination due to oligodendrocyte dysfunction. (Kang et al., 2013). Another research data reported reductions in MBP content in the spinal cords of post-mortemed ALS patients (Pons et al.;.
In the present study, long-term exposure to 5 mg/kg MeHg via oral route resulted in marked alterations in MBP content in brain homogenates and CSF rats. Treatment with 4-HI, on the other hand, resulted in the steady, dose-dependent restoration of impaired MBP content in respective biological samples. Based on the aforementioned data, it is possible to infer that 4-HI may prevent myelin sheath degeneration, white matter destruction, and demyelination caused by MeHg, and have the potential to initiate the remyelination process. These ndings are noteworthy in terms of understanding the potential relationship between 4-HI and neuropathogenic processes. However, more research into the molecular processes behind such neuroprotective activities is required.
Several neuropathognomonic mechanisms, such as glutamate-mediated excitotoxicity and oxidative stress, can disrupt neuronal homeostasis, eventually activate the cellular apoptotic cascades, ultimately leading to motor neuron death . According to our ndings, pro-apoptotic markers such as caspase-3 and Bax increased progressively in the brain homogenate and blood plasma of MeHgtreated ALS-like rats. A distinct trend was observed with the anti-apoptotic protein, and it was found that Bcl-2 levels markedly decreased after 21 days of continuous oral MeHg administration. However, after long-term treatment with 4-HI, the levels of these apoptotic markers in rat brain homogenate and blood plasma were observed to be reversed in a dose-dependent manner. Such intriguing ndings imply that 4-HI may be capable of preventing the increasing death of motor neurons and thereby combat ALS progression.
Accumulating evidence suggests that glutamate excitotoxicity may be implicated in the neurodegenerative presentations of ALS (Dong et al., 2021, Gaywali et al., 2021. Imbalances in excitatory and inhibitory neurotransmitters may have a role in developing ALS and related brain dysfunctions (Cheng et al., 2021, Pradhan et al., 2021. Glutamate excitotoxicity has been linked to oligodendrocyte death and may cause white matter degeneration in the spinal cord (Park et al., 2004). An increase in glutamine levels (an excitatory mediator) has been reported in the motor cortex and white matter of patients with ALS, compared with the healthy controls (Foerster et al., 2013). In the current study, the level of various neurochemicals in the rat brain homogenate was quanti ed by ELISA. MeHg exposure remarkably decreased GABA, acetylcholine, and serotonin levels, while considerably raising glutamate levels, indicating glutamate-mediated excitotoxic effects. The administration of 4-HI substantially restored the altered concentrations of these brain neurotransmitters in a dose-dependent manner.
Neuronal in ammation has been identi ed as a prominent pathological hallmark in ALS, and cytokineinduced in ammation may play a key role in ALS pathology. According to research, MeHg increases the production of various in ammatory cytokines such as TNF-α, IL-1β, and IL-6 in brain tissue and neural stem cells, which in turn triggers neuroin ammation and induces selective CNS damage (Shimada et al., 2016). A study found signi cant levels of in ammatory cytokines and chemokines in plasma samples from ALS patients than in controls. The study also revealed that neuromuscular dysfunction in ALS involves systemic regulation of in ammatory markers . Another research rea rmed the role of in ammation in ALS, nding that serum TNF-α and IFN-γ levels were considerably higher in ALS patients compared to normal subjects (Babu et al., 2008).
To address such interesting evidence from multiple studies, we investigated the anti-in ammatory effect of 4-HI treatment on TNF-α and IL-1β levels in ALS-like rats via ELISA. Rats treated with MeHg exhibited an increase in the concentration of these in ammatory mediators and consequently indicated evidence of in ammation. However, our observations suggest that chronic oral treatment of 4-HI dose-dependently reversed the altered in ammatory cytokine levels in rat brain homogenate and blood plasma.
Previous studies have also shown that MeHg exposure triggers an oxidative stress response in the neurons, resulting in increased ROS generation and oxidative phosphorylation (Limke et al., 2004).
The current study aims to determine 4-HI's antioxidant and neuroprotective effects on the generation of oxidative stress biomarkers. The induction of an ALS-like phenotype in rats resulted in a marked increase in oxidative stress indices such as AchE, LDH, MDA, and nitrite. In contrast, antioxidants such as SOD and GSH were considerably decreased. Long-term treatment of 4-HI substantially lowered the levels of these oxidative products while increasing the concentration of antioxidants like SOD and GSH in whole-brain homogenate fractions. This suggests that 4-HI may play an antioxidant role in reducing the risk of oxidative stress.
Numerous studies have a closely correlated loss of myelin in the white matter tracts and oligodendroglial involvements to ALS onset (Saberi et al., 2015). A morphometric investigation was carried out to detect volumetric differences in the whole brain. It was shown that ALS patients had a decrease in whole-brain volume compared to healthy control subjects (Mezzapessa et al., 2007). Another research revealed a substantial reduction in white matter volume in ALS patients than controls (Abrahams et al., 2005). A similar study found considerable white matter atrophy among the ALS subgroups (Ellis et al., 2001).
In the present study, morphological, gross pathological, and demyelination volume were assessed, following the ALS-like phenotype in rat brains. Prolonged 4-HI therapy exhibited neuroprotective potential against MeHg-induced gross morphological and pathological alterations. Our ndings revealed that 4-HI therapy effectively repaired gross morphological defects in the whole brain and signi cantly reduced the pathogenic alterations in brain sections. Additionally, 4-HI modulated the level of MBP in damaged neurons. Furthermore, 4-HI demonstrated a signi cant, dose-dependent improvement in myelin restoration and reduction in demyelination volume. These ndings show that chronic administration of 4-HI could be a promising pharmacological strategy for treating demyelinating disorders such as ALS.
However, the current ndings are only associations in which 4-HI was principally investigated to alleviate neurobehavioral and neurochemical abnormalities in MeHg-treated ALS-like rats by modulating or interfering with the IGF-1/GLP-1 signaling. Based on the current ndings, it can be suggested that the levels of IGF-1, GLP-1, and MBP in brain tissue, blood plasma, and CSF can be used as effective and reliable early diagnostic biomarkers to predict an important degenerative component of the ALS brain.
Above all, through knock-in/knock-out studies of the IGF-1 and GLP-1 genes, a mechanistic approach must be veri ed. Furthermore, molecular support of this hypothesis is necessary by correlative investigations such as Western Blot and immuno-histochemistry for the cellular markers. Despite these study limitations, the strength of the discovered neuroprotective bene t of 4-HI in restoring or upregulating the altered state of IGF-1/GLP-1 signaling cascades in the brain may lead to the development of a much-needed, novel, disease-modifying therapeutic intervention against this neurodegenerative disease.

Conclusion
Insights from our experimental results reveal that our protocol drug, 4-HI, interacts with IGF-1/GLP-1 signaling and in uences various pathological alterations. Indeed, no pre-clinical investigations on the neuroprotective impact of 4-HI via the dual activation of IGF-1 and GLP-1 signaling in MeHg-treated ALSlike rats have been undertaken to date. Therefore, we may infer from our ndings that 4-HI could be a promising therapeutic drug candidate for treating a range of behavioral, molecular, neurochemical, and gross morphological changes associated with the development and progression of the ALS-like phenotype. This study also examined the levels of apoptotic proteins like caspase-3, Bax, Bcl-2, and neuroin ammatory cytokines like TNF-α and IL-1β in rat blood plasma. Interestingly, our ndings show that 4-HI stimulates neuronal proliferation, growth, and differentiation inhibits apoptosis and increases myelin-basic protein levels in MeHg treated ALS-like rats via positively regulating the IGF-1/GLP-1 pathways in the rat brain. Furthermore, the effective restoration of pathological and gross morphological anomalies in the whole brain and brain sections demonstrates 4-HI's neuroprotective ability against MeHg-induced neurological defects.
Henceforth, these fascinating ndings can be utilized as potential diagnostic biomarkers in the future to search for a disease-modifying pharmacological moiety at a preliminary stage. However, further genetic studies and immuno-histological assessments are required to characterize the underlying mechanisms governing such interactions. Indeed, we can compare the IGF-1 and GLP-1 as futuristic therapeutic targets to other conventional drug therapies. Cysteine-aspartic proteases, cysteine aspartases or cysteine-dependent aspartate-directed proteases-3 CNS Effect of 4-hydroxyisoleucine on gross pathological changes (brain sections) in methylmercury-induced ALS-like rats (a) Normal control (i. Cerebral cortex ii. Hippocampus iii. Basal ganglia) (b) Vehicle control (c) 4-HI100 perse (d) MeHg5 (e) MeHg + 4-HI50 (f) MeHg + 4-HI100 (Scale bar: 5 mm) Note: Yellow circles are pointing to the demyelinated area.