INTRODUCTION:

The incidence of cardiac arrest ranges from 0.04 - 0.13% of the total population per year. The number of patients with a good outcome after discharge from the hospital is only in the range of 11-48%¹. The prevalence of cardiac arrest outside the hospital in the United States, which can survive and living normally without brain damage, is less than 10%^2,3.

The cause of death in patients who achieve ROSC (Return of Spontaneous Circulation) is mostly due to nerve or brain damage².Cessation of cerebral circulation after cardiac arrest causes neuronal ischemia. Damage to neurons due to ischemia can occur rapidly, especially in areas of the brain that are sensitive to ischemia such as the claustrum of the cerebral cortex, CA1 region of the hippocampus and purkinje cells in the cerebellum⁴.

Hypothermia as an intervention in neuroprotection efforts has been shown in many reports to effectively maintain tissue and limit injury after various acute brain tissue ischemias^5,6. The International Liasion Committee on Resuscitation (ILCOR) has recommended hypothermia therapy in anyone who is in a coma or ROSC due to Ventricular Fibrillation, Ventricular Tachycardia, or other rhythm disorders⁷. Therapeutichypothermia can be conducted in several ways. Forced hypothermia (physical) uses an external approach with an ice pack or cold blanket, as well as an internal approach using a special central venous catheter⁸, or by cold intravenous infusion^9-11. The forced hypothermic (physical) method is considered inefficient in lowering the core temperature of adult subjects due to the naturally occurring thermoregulatory response such as shivering, cutaneous vasoconstriction and increased metabolism¹². The regulated hypothermia technique using HBN-1 (Hybernation-1) was developed with the hope of being able to answer and become a pharmacological alternative to the therapy of hypothermia through pharmacological agents. HBN-1 is a patented pharmaceutical preparation consisting of a mixture of ethanol, vasopressin and lidocaine^12,13. The study aimed to analyze the neurobehavior function improvement in the hypothermia regulated therapy group using HBN-1 compared to the forced hypothermia (physical) method (ice pack), on animal model.

MATERIAL AND METHODS:

This is a true experimental study, with a post-test-controlled group design. This study used male rats (Wistar) obtained from a provider institution with standard qualifications and a good reputation. Before getting the research treatment, screening was carried out with several criteria. The inclusion criteria included body weight of Wistar rats, 200-350 grams, male, healthy, and 5 months of age. The exclusion criteria included difficult intubation, more than 30 minutes of not being intubated, resuscitation up to 2 minutes without ROSC, and the sample was not representative to be examined.After going through screening with inclusion and exclusion criteria, randomization was carried out (random allocation). As many as 16 animals were divided into 4 groups, namely the non-treated group (control group), normothermic treatment group, forced hypothermia treatment group (physical), and regulated hypothermia treatment group (HBN-1).

In this study, two types of designs were obtained, designs for biochemical and neurobehaviour examinations. The total number of treatment groups, there are 7 groups: K1 group: no treatment group (control group); Group P1: normothermia treatment group then biochemical analysis was performed after decapitation; Group P2: normothermia treatment group then analyzed for neurobehaviour outcome for 7 days then decapitation and biochemical analysis were performed; Group P3: forced hypothermia (ice pack) treatment group, biochemical analysis was performed after decapitation; Group P4: forced hypothermia (ice pack) treatment group, neurobehaviour outcome analysis was performed for 7 days then decapitation and biochemical analysis were performed; Group P5: hypothermic treatment group with HBN-1 then biochemical analysis was performed after decapitation; Group P6: hypothermia treatment group with HBN-1 then analyzed for neurobehaviour outcomes for 7 days then decapitation and biochemical analysis were performed

Data Collection Procedure:

Rats were premedicated by injection of xylazine 5 mg/kgBW, followed by anesthesia with ketamine 50 mg/kgBW and administered normal saline infusion of 100cc/kg BW/24 hours, installation of an arterial line catheter (to monitor arterial blood pressure, as well as ensuring the condition of the cessation of the artery pulsation in cardiac arrest state), monitoring the ECG, rectal and esophageal temperature, and shaving the hair on the chest area and parts of the right and left thighs. Then the rats were intubated with 14G intravenous catheter. Maintenance with inhalation anesthetics using isoflurane. Cardiac arrest was carried out by inducing ventricular fibrillation for 3 minutes using an AC power supply with a voltage of 12 voltage through 2 electrodes in the form of a needle placed in the apex area of the heart and the right arm of the rat. Hemodynamic changes in cardiac arrest markers are monitored through changes in heart rhythm to ventricular fibrillation or loss of pulsation from arteries on a monitor that occurs for more than 30 seconds. Resuscitation was performed until the ROSC was reached within 2 minutes.

After ROSC was achieved, the rats were randomly divided into groups. The normothermic group rats were left at a normal temperature of 36.8ºC for 3 hours. The rats in the forced hypothermic group were immediately cooled with ice packs until the rectal and esophageal temperature of 33 -35.9 °C was reached, and kept at this temperature for 3 hours. HBN-1 group was cooled by injecting HBN-1 bolus 20 mL/kgBW for 60 minutes with ambient (environment) temperature of 28-32 °C followed by infusion of HBN-1 with dose of 3 mL/kgBW/hour for 3 hours. The treatment group was returned to the cage and received standard rat food and drink for 7 days, beforeconduct the neurobehaviour evaluation.

The neurobehaviour outcome analysis will be assessed using the Rat ND score. This parameter has been used previously by Katz et al (1995), specifically in rats' cardiac arrest model¹⁴. This scoring includes sensory-motor examinations (grooming, forepaw grasp, limb movement, and limb sensation), and assessment of global awareness, respiratory function, cranial nerve function, body axis tone, righting¸ locomotion, and balance. The Rat ND score ranged from 0 (no neurobehavior deficit) to 100 (very severe neurobehavior deficit).After evaluation, euthanasia was carried out under deepened anesthesia with xylazine bolus 5mg/kgBWintravenouslywith a combination ofketamine 50 mg/ kgBWintravenously, then the rats were euthanized by decapitation.

RESULTS:

Time Needed to Reach Hypothermic Conditions:

The results of the Shapiro-Wilk normality test showed that the HBN-1 group was not normally distributed (Sig. <0.05), so the statistical test was carried out with Kruskal Wallis. The mean rank showed that the fastest method to achieve hypothermia was force hypothermia group, with the smallest Mean Rank. The Kruskal Wallis test results showed a significant difference between the force hypothermia group and the regulated hypothermia (HBN-1), with p value = 0.010 (p <0.05).

Outcome of Neurobehaviour Function:

On the Rat ND Score variable, the analysis was performed using the Kruskal Wallis test. The aim of this analysis was to determine whether there were differences between groups regarding the most severe symptoms of neuro-deficit (The greater the mean rank, the greater Rat ND score). Significance value <0.05, indicating a significant difference between groups.Analysis on the Rat ND Score component of the assessment showed a significant difference in the treatment group with normothermia. Rats treated in normothermic conditions showed the highest deficit score (highest mean rank) in the olfactory component (p = 0.046), vision (p = 0.037), hearing (p = 0.010), motor deficit (p = 0.032), and sensory deficit (p = 0.05). The olfactory component was assessed by the behavior of rats in smelling and detecting food odors. The hearing component was assessed by the behavior of the rats that responded to the clapping sound of the researchers. The motoric deficit component was assessed from the movement of the rats' legs and tail, whether they were normal, stiff, or limp. The sensory deficit component was assessed by the presence or absence of the response of the rats' feet and/ or tails when given stimuli in the form of pinching. The findings of differences in the normothemia group were followed by the Mann Whitney test.

The data was then performed using non-parametric analysis related to the abnormal distribution of the data. The data distribution was not normal because there were lots of constant data with a value of 0. Based on the Mann Whitney continued test, it was found that the regulated hypothermia group showed no significant difference in the total Rat ND Score against the control group, p value= 0.139 (p> 0.05), which indicates low total Rat ND Score (no neurological deficits). The normothermic treatment group showed a significant difference (p <0.05) against the control group, and hypothermia (force hypothermia, and HBN-1) related to the total Rat ND Score, as well as some of its components (olfactory, vision, hearing, motor deficit, and sensory deficit.In each parameter/component of the Rat ND score, the Friedman test was also carried out to analyze the difference in the Rat ND Score between parameters. The results of the Friedman test showed that there were differences between several parameters of the neuro-deficit component. The highest score reflects the most severe condition, which is the sensory deficit component. Whereas the least or the least common are the whisker components and the function of respiration.

Experimental mice in groups P2, P4, P6 were kept alive for up to 7 days, then compared the neurobehavior outcomes per day from each group (HBN-1, forced hypothermia, and normothermia). Analysis of the neurobehaviour component was carried out by assessing the Rat Neuro-Deficit (ND) score. Rat ND score is a system to measure the neurobehavior function of rats. This scoring includes a sensory-motor examination (grooming, forepaw grasp, limb movement, and limb sensation), as well as an assessment of global awareness, respiratory function, cranial nerve function, body axis tone, righting¸ locomotion, and balance. Rat ND scores ranged from 0 (no neurobehaviour deficit) to 100 (very severe neurobehaviour deficit).

Total Rat ND Score was evaluated in each group for 7 days of assessment. The trend of decreasing Rat ND Score is seen every day, from each group. The HBN-1 group (P6) tended to show a lower total Rat ND Score than forced hypothermia, on the 1st, 2nd day, and a score of 0 was obtained since the 3rd day after hypothermia treatment. A score of 0 indicates the absence of neurological disorders or deficits in mice. Meanwhile, forced hypothermia showed a very low Rat ND score but was still detected from day 3 to 7 with a stable level.The bar chart shows the parameters with the highest scores being sensory deficit, followed by motor deficit, hearing, vision, and olfactory. A high score indicates a degree of neurological deficit. The normothermia group (P2) showed higher neurological deficits than the other groups.On the Rat ND Score, the analysis was carried out using the Kruskal Wallis test to determine differences in neuro-deficit between groups. The greater the mean rank, the greater the Rat ND score. In the Kruskal Wallis test table, the highest total Rat ND Score was found in the P2 (normothermic) group with a mean rank of 13.75, and p=0.012.

The data was then carried out using a non-parametric test related to the distribution of the data that was not normal, which was due to a lot of data being constant with a value of 0. Based on the Mann Whitney follow-up test, the treatment group with normothermia (P2) showed a significant difference (p<0.05) to the control group (K1 ), and hypothermia (P4, P6) related to the total Rat ND Score, as well as some of its components (olfactory, vision, hearing, motor deficit, and sensory deficit). The olfactory component shows the behavior of mice in detecting food odors. The hearing component shows the behavior of mice that respond to the sound of clapping hands. The motor deficit component was assessed from the movement of the rat's legs and tail. The sensory deficit component was assessed from the presence or absence of the response of the rat's feet and/or tail when given a pinching stimulus. The lowest p-value reflects the most severe condition, namely Sensory Deficit (p=0.005), which is most affected by ischemia – cardiac arrest.In the follow-up analysis of Mann Whitney the regulated hypothermia group (P6) showed no significant difference in the total Rat ND Score against the forced hypothermia group (P4), with p value = 0.083 (p>0.05). However, in component analysis, regulated hypothermia with HBN-1 (P6) showed a significantly lower score than forced hypothermia, on motor deficit (p=0.046), and sensory deficit (p=0.015).

Correlation of Rat ND Score with Claustrum Neuron Cell Apoptosis Rate:

The level of neurological deficit (Rat ND Score), then analyzed related to neuronal cell apoptosis in the claustrum area. Normality test was carried out on Rat ND Score data, and the level of apoptosis of claustrum area neuron cells. The Shapiro-Wilk test was used because the sample size was <50. The analysis showed an abnormal distribution on the Rat ND Score (p=0.000) and apoptosis (p=0.007). Furthermore, non-paramaterial statistical tests were carried out using the Spearman correlation test.

Spearman's test showed a significant relationship between Rat ND Score and the level of neuronal cell apoptosis in the claustrum area (p=0.000), with a correlation coefficient of 0.843. The correlation coefficient > 0.8 indicates a high correlation level, and a positive value indicates the higher the Rat ND Score, the greater the number of neuron cells in the cerebral claustrum area undergoing apoptosis.After confirming that there is a relationship between Rat ND Score and apoptosis, the coefficient of determination and linear function in the graph are calculated. Linear graph analysis was used to further estimate the correlation between the two variables in the study (Rat ND Score and claustrum neuron cell apoptosis). The coefficient of determination (R2) was obtained at 0.6575, so that the Rat ND Score and cell apoptosis variables were stated to be well correlated because R2>0.5. The coefficient of determination (R2) obtained is 0.6575, so the magnitude of the change in apoptosis by the Rat ND Score variable is 65.75% while the rest is caused by other factors.

DISCUSSION:

In our study, the cardiac arrest of the rats was seen from electrocardiogram waves with characteristic of ventricle fibrillation (VF), plus confirmation of arterial pulsation loss. Previous large-scale studiesshowed a significant reduction in mortality within six months in patients who were given mild therapeutic hypothermia, which included only patients with VF/VT as the initial heart rhythm¹⁵.The normal body temperature in healthy individuals (as measured in the oral cavity) is 36.8 +/- 0.4°C with variation of 0.5°C. Rectal temperature is commonly 0.4°C higher than oral temperature. Rectal temperature more closely reflects core temperature, although temperature measured through a pulmonary artery catheter correlated most closely with brain temperature. The approach with rectal temperature measurement was chosen because of its convenience, although the pulmonary artery temperature is considered the gold standard in anesthesia and in intensive care units. Other methods, for example tympanic temperature measurement, do not allow continuous temperature monitoring¹⁶. For most purposes, rectal temperature is considered the most practical and accurate measurement; However, the accuracy of this method still behind esophageal temperature¹⁷. Rectal temperature measurement may be affected by stool, and the exact position of the probe¹⁶.

In our study, hypothermia is defined as the body temperature of experimental animals in the range of 33-35.9°C. In clinical practice, temperatures of 33°C - 36°C are generally classified as mild hypothermia, 28°C - 32°C as moderate hypothermia, and below 28°C as deep hypothermia¹⁸. Hypothermia induction was carried out in two approaches, namely force hypothermia (physical) using an ice pack, and regulated hypothermia using HBN-1. Force hypothermia in our study showed a shorter time to achieve hypothermia than HBN-1. However, our study is the first experimental study to analyze the performance between HBN-1 and ice packs.The previous study by Katz et al., (2015), examining regulated hypothermia (HBN-1) vs force hypothermia, was used 4 °C saline infusion. The study stated that HBN-1 shortened the time (85 ± 71 minutes) to target temperature (33.5 ° C) versus physical hypothermia (247 ± 142 minutes), with (p <0.0001). Several factors in our study are thought to cause HBN-1 to reach temperature longer than force hypothermia (ice pack). The first factor was the use of HBN-1 dose of 20 ml/kg, compared to studies byKatz et al., (2012)using a HBN-1 dose of 30 ml/kgBW. Ethanol in HBN-1 is responsible compound for the hypothermic process. Ethanol works by lowering the core temperature and triggering hypothermia in the center of body temperature regulation in the hypothalamus. When the core temperature drops, the body responds by reducing the metabolic rate, inhibiting the shivering reflex, and inducing heat loss through peripheral vasodilation¹⁹.The second factor is the use of inhalation anesthetics in our procedure. The use of inhalation anesthetics triggers vasodilation and increases the effect of blood flow and changes in metabolic patterns²⁰. Vasodilation conditions will increase the surface area of blood vessels, and increase the temperature transmission of the ice pack which in turn accelerates the induction of hypothermia. The third factor is the influence of ambient temperature. Based on the literature, the effect of ethanol on body temperature is poikilothermic (depending on the environment/ambient temperature in which the animal is tested)²¹. In our study, the ambient temperature was between 28-32°C, while research byKatz et al., (2012) uses the ambient temperature in the range of 19 °C. This difference in environmental temperature is thought to play an important role in the time needed of hypothermia induction using HBN-1. Heat loss occurs in five ways: radiation (40%), convection (30%), evaporation (15%), conduction (5%), and respiration (10%). The radiation, convection, and evaporation mechanisms can be affected by ambient temperature²².

In our study, hypothermia was maintained for up to 3 hours. This approach refers to studies that show optimal mild hypothermia (34 ºC for around 4.5 hours), which reduces intracellular calcium, ROS, and restores mitochondrial transmembrane potential. The optimal therapeutic hypothermia strategy for severe hypoxic-ischemic neurons, of a certain duration may exert a neuroprotective effect, and that therapeutic hypothermia may initiate neuroprotective effects by inhibiting the mitochondrial apoptotic pathway²³.The evaluation of neurological function was carried on the 7th day after treatment. Our study refers on studies byChe et al., (2011), who investigated the effects of therapeutic hypothermia on survival and neurological function of experimental animals Long Evan Rats. Outcomes evaluated were included 7-day survival, 7-day survival with good neurologic function, and selective survival of susceptible hippocampal CA1 sector pyramidal neurons. Neurological function scores were measured daily²⁴.Our study show that the Rat ND Score in the hypothermia group was significantly higher than the normothermic group. However, the regulated hypothermia group (HBN-1) showed no statistically difference against the force hypothermia group, in the total Rat ND Score. However, the neurobehavior function of the regulated hypothermia group (HBN-1) tends to be better (the average Rat ND Score is lower than forced hypothermia).

Based on experimental reports, there have been a variety of beneficial histological, biochemical, and pathophysiological effects reported on neuron cells associated with hypothermia. These beneficial effects include decreased levels of the excitotoxic metabolite glutamate in the cerebrospinal fluid, reduced vasogenic edema at the site of injury, decreased neutrophil invasion²⁵. There is also evidence that hypothermia decreases tissue metabolism and energy requirements, oxidative stress, tissue bleeding, and apoptosis²⁶.The neuroprotective effects of hypothermia are mainly associated with decreased metabolic rate and decreased cerebral blood flow. Hypothermia decreases the brain's metabolic rate from oxygen consumption and glucose metabolism. Hypothermia can suppress the final consequence of increased lactate production due to dependence on anaerobic metabolism and the development of acidosis by maintaining brain metabolic stores. Hypothermia maintains high-energy phosphate compounds, such as adenosine triphosphate (ATP), and maintains tissue pH, all mechanisms that may be associated with a decreased effect on brain metabolism, which maintains tissue ATP levels. ATP is required to maintain the ion gradient, and when this concentration gradient is disturbed, as in the case of ischemic stroke, calcium ingests and leads to an increase in extracellular glutamate levels. Hypothermia also significantly decreases excitotoxin release and calcium influx due to cerebral ischemia²⁶.

Experimental evidence and clinical studies suggest that hypothermia affects nearly every metabolic, molecular, and cellular event in cell death to increase tissue bioavailability. A study that reviewed various methods of therapeutic hypothermia (both classical hypothermia methods, and pharmacological induction of hypothermia) similarly demonstrated that the goals of neuroprotection are achieved, with neuroprotection requiring a multi-targeted approach. Hypothermia also expands the therapeutic window for the management of nerve cell protection, regarding combination therapy with other neuroprotectant drugs²⁶.The effect of hypothermia on HBN-1 is mainly due to the ethanol content. In our study, the ethanol dose in HBN-1 has gone through an adjustment process, refers toKatz et al., (2015), so it is not included in the toxic dose. In a study that tested low-moderate doses in rats, it showed an acceleration of neurological development such as: walking, vertical screen grasp, and balance when walking on a thin rope. This finding considers two alternative explanations. First, ethanol can directly improve neurobehavioral outcomes at certain doses. Second, ethanol can be protective against the possible effects of stress²⁷. The neuroprotectant effect of ethanol is amplified by the hypothermic effect it produces, because hypothermia has a strong neuroprotectant effect on cardiac arrest and cerebral ischemia. In this case, the neuroprotection mechanism appears to be partly due to a decrease in metabolism in the brain²⁸. The improvement in ethanol-related neurobehaviour needs to be interpreted with caution.

The vasopressin content in the HBN-1 also played an important role in the suppressing the neurological deficit. Vasopressin modulates nerve function by acting as neurotransmitter. Vasopressin prevents deprivation-induced programmed cell death (apoptosis) in the neuronal cell line, which expresses endogenous V1 receptors. Vasopressin exerts this protective effect through activation of protein kinase C alpha and beta, Erk/ribosomal S6 kinase (RSK) MAP kinase pathway, and PI3 kinase/Akt pathways. This mechanism strongly indicates that vasopressin has neuroprotective characteristics in the brain tissue. The results of studies on rat hippocampal neuron cultures showed that activation of endogenous V1 receptors by vasopressin protected hippocampal neurons against glutamate-induced deprivation and neuronal cell death²⁹. The literature also confirms the neuroprotective effects of lidocaine(as component of HBN-1). Previous animal studies have shown that lidocaine is neuroprotective against hypoxia and ischemia. The underlying mechanism is unclear and is thought to be multifactorial, including inhibition of influx sodium, preservation of cellular mitochondria, maintaining ATP, and suppressing inflammatory processes in neuron cells³⁰. Other studies suggest lidocaine provides brain protection through several mechanisms, including reducing brain metabolic rate, reducing ischemic excitotoxin release, slowing ischemic transmembrane ion shift, anti-inflammatory, and anti-apoptotic properties³¹.

Analysis of the components of the Rat ND Score assessment in our study showed that there were significant differences in the treatment group with normothermia in the olfactory, vision, hearing, motoric deficit and sensory deficit components. This finding relates to ischemia occurring in selectively vulnerable neurons (SVN). Novel literature stated that the SVN located in the claustrum of the cerebral cortex, CA1 pyramidal neurons in the hippocampus, purkinje cells of the cerebellum, and other brain regions including the neocortical area, caudate nucleus, globus pallidum, putamen, thalamus reticular nucleus. These regions have their respective roles and can be related to one another³². Claustrum has an important role in connecting almost all areas in the brain, especially in regulating consciousness³³.Claustrum serves as a conductor for certain aspects of advanced integration of brain activity, the integration of auditory and visual signals also involves coordination by other areas including the parietal cortex and the pulvinar. Inhibition or disruption of the claustral area suggests an impact on large-scale cortical coordination. The literature predicts disruption in the claustrum with respect to activity in the somatosensory, vestibular, and other cortical area functions³⁴.The hippocampus is mainly composed of pyramidal cells, which play a role in afferent and efferent processes. The hippocampus is known not only to be important in learning and memory processes but also in spatial navigation, emotional behavior, and regulation of hypothalamic function. In the innervation pathway, the hippocampus receives input from the temporal association cortex via the perirhinal and entorhinal areas to CA1 neurons. The hippocampus is also part of the ventral striatal loop, so it can influence motor behavior. Although emotional behavior is regulated primarily by the amygdala, the hippocampus and amygdala are both interrelated, so they can influence one another³⁵.

Disruption or damage to purkinje's cerebellar cells can cause ataxia which manifests as disturbances in gait, balance, and axial instability³⁶. Loss of Purkinje cells after cardiac arrest may contribute to neurological dysfunction, including post hypoxic myoclonus³⁷. Other brain regions include the neocortical layer, caudate nucleus, globus pallidum, putamen, and thalamic reticular nucleusalso play several roles in the manifestation of disorders such as olfactory function. In thebasal ganglia, which is a striatal complex, includes the corpus striatum (caudatus and putamen), also interconnected with afferent projections (globus pallidus)³⁸.

Outcome Neurobehaviour

Cognitive deficits are a common problem in patients with cardiac arrest who are successfully resuscitated. These mechanisms lead to global brain ischemia, and previous pathological studies have revealed the detrimental effects of hypoxia-ischemia³⁹.There were several groups of experimental animals in this study whose neurobehavior function was evaluated every day for 7 days after treatment. This parameter was measured by the Rat ND score (Rat Neuro-Deficit Score), which is a scoring system to measure the neurobehavior of rats. This scoring includes a sensory-motor examination (grooming, forepaw grasp, limb movement, and limb sensation), as well as an assessment of global awareness, respiratory function, cranial nerve function, body axis tone, righting¸ locomotion, and balance function. The higher the Rat ND-Score, the more severe the neurobehavior deficit in experimental animals.

This research refers to a study byChe et al., (2011), who investigated the effects of therapeutic hypothermia on survival and neurological function in Long Evan Rats experimental animals. In this study, the outcomes evaluated included 7-day survival, 7-day survival with neurological function as measured by the Neurological Function Score (NFS). Neurological function scores were measured daily for 7 days[24]. The evaluation period for the neurological function of Long Evan rats in 7 days was used as a reference for the duration of observation. However, the use of the Rat ND Score instrument refers to the study by Katz et al (1995) using Wistar strain mice, as was the case in this study.In this result, the largest total ND Score (highest level of neurological deficit) was found in the normothermic treatment group, with significant differences from other groups.The high neurological deficit in the normothermia group in this study was also confirmed by several other studies. A study in normothermic rats demonstrated neurobehavioral deficits in the learning paradigm after cardiac arrest. Post-cardiac arrest spatial memory deficits in rats and mice have also been reported. Mice after cardiac arrest under normothermic conditions (without intervention), were indeed associated with a reduction in the total distance traveled in the open field test, and a decrease in time spent in the light compartment in the light/dark preference test. In the study, behaviors assessed included awareness, interaction, ability to pick up the wires, motor function, and overall activity level⁴⁰. Other studies also suggest a decrease in learning and memory functions, related to hypoxic-ischemic brain injury after cardiac arrest⁴¹.

In the setting of hypothermia, clinical evidence suggests that prolonged-moderate cerebral hypothermia that begins within hours of severe hypoxia-ischemia and continues until resolution of the acute phase of delayed cell death can reduce further neuronal damage and improve behavioral recovery in term infants and adult patients. after cardiac arrest⁴².The results of further analysis on the parameters of the Rat ND Score examination in this study showed that the parameter with the highest score was sensory deficit, followed by motor deficit, hearing, vision, and olfactory. A high score indicates the degree of neurologic deficit in these parameters. Neurological deficits that occur are thought to be related to damage to various regions of the brain that have specific functions.Huang et al., (2017), mapped areas in the brain that are most prone to ischemic processes, including: the claustrum in the cerebral cortex, CA1 pyramidal neurons in the hippocampus, Purkinje cells from the cerebellum, and other brain regions (3rd to 6th neocortical layers, caudate nucleus, globus pallidum, putamen, nucleus reticular thalamus).

Claustrum has an important role in connecting almost all areas of the brain, especially in regulating consciousness³³.Other literature states that the claustrum interacts with almost all other brain structures: motor cortex, somatosensory cortex, prefrontal cortex, cingulate cortex, auditory and visual cortex, hippocampus, amygdala, and caudate nucleus⁴³.The clatrum serves as a conductor for certain aspects of advanced integration of brain activity, integration of auditory and visual signals also involves coordination by other areas including the parietal and pulvinar cortex. Inhibition or disturbance of the claustrum area suggests a large-scale impact on cortical coordination. The literature suggests disturbances in the claustrum are related to activity in somatosensory, vestibular, and other cortical area functions³⁴. The theories in the literature are in accordance with the findings of neurological deficits in this study, where the highest Rat ND Score was obtained on the parameters of sensory deficit, motor deficit, hearing (auditory), and vision (visual).

Meanwhile, the olfactory deficit parameter, which is also significant in this study, is thought to be related to disorders in other susceptible brain regions. Other brain regions, including the neocortical layer, caudate nucleus, globus pallidum, putamen, reticular nucleus of the thalamus, also play a role in the manifestation of ischemic disorders in the brain³². The nucleus accumbens as part of the olfactory system, together with the olfactory tubercle, is the medial extension of the caudate-putamen³⁸. This study did show that the Rat ND Score in the hypo-acceptance group was significantly higher in the normothermia treatment group. However, statistically the regulated hypothermia group (HBN-1) showed no difference between the total Rat ND Score and the forced hypothermia group. Although not significantly different, in the regulated hypothermia (HBN-1) group, neurobehavior function tended to be better (average Rat ND Score was lower than forced hypothermia). This finding was associated with better neuroprotection parameters seen from the results of other biomarker studies (increased HSP70, increased catalase, decreased caspase-3, and decreased F2-Isoprostane). As in the study by Yu et.al.,in experimental mice⁴⁴, the number of cells expressing caspase-3 in the sub-granular zone of the injured hemisphere correlated well with the neurogical deficit score in the hypothermic group.

The level of Rat ND Score in the HBN-1 group which tends to be lower (as seen from the daily score which has reached 0 on the 3rd day), is thought to be related to the ethanol content as the main component of HBN-1. The beneficial effects of ethanol on neurological function were reported in a study that tested low (2.4 g/kgBW) to moderate (2.9 g/kgBW) doses in rats. The results of this study indicate an acceleration of neurological development such as: walking, vertical screen grasp, and balance when walking on a thin rope²⁷. However, other literature suggests a prolonged adverse effect on neurobehavioral development at high ethanol doses, especially when confirmed by high blood ethanol concentrations. Pathophysiologically, ethanol-associated toxic dose exposure is associated with neuronal loss in cortical regions. The mechanism associated with ethanol-induced neuronal death is a consequence of increased oxidative stress and induction of pro-inflammatory mediators including cytokines, COX-2 and iNOS⁴⁵.Ultimately, these findings lead to two alternative explanations. First, ethanol can directly improve neurobehavioral outcomes at certain doses. In this study, the dose of ethanol in HBN-1 was adjusted according to theKatz et al., (2015)so it is not included in the toxic dose. Second, ethanol can be protective against the possible effects of stress. In these cases, the beneficial effects on neurobehavior associated with ethanol administration need to be interpreted with caution.

Correlation of Rat ND Score and Neuron Cell Apoptosis Rate in Claustrum:

In this study, the analysis focused on biomarkers in the claustrum cerebri, which is an area that has not been widely discussed in previous studies. The findings on these biomarkers were then linked to the overall neurological outcome (level of neurologic deficit), to a per-component analysis. Analysis of the components of the assessment of the Rat ND Score showed significant differences in the treatment group with normothermia in the olfactory, vision, hearing, motor deficit, and sensory deficit components. While the components of consciousness, respiration, corneal, whisker, travel edge, placing test, righting reflex, and stop at the edge of the table, did not show significant differences. These findings relate to ischemia that occurs in selectively vulnerable brain regions (SVN), which are located in the claustrum of the cerebral cortex, CA1 pyramidal neurons in the hippocampus, Purkinje cells of the cerebellum, and other brain regions including the 3rd to 3rd neocortical layers. 6 caudate nucleus, globus pallidum, putamen, reticular nucleus of the thalamus. These regions have their respective roles and can be related to each other³².

Based on the existing literature, markers of neuronal cell damage in the claustrum have not been studied more thoroughly before, especially as an area that shows selective susceptibility. However, recent studies have shown the claustrum is the gray matter structure most susceptible to neuronal ischemic changes in cardiac arrest[33].Rat Neuro-Deficit (ND) Score introduced byKatz et al., (1995), which is a scoring system modified from the Dog ND Score. The total Rat ND score consists of components: awareness and breathing. cranial nerve function, motor function. sensory function, and coordination. Normal mice have an ND score of 0% and brain death mice have an ND score of 100%.As shown on a map of the rat's central nervous system byBruguier et al., (2020), schematic features of the connectivity of the claustrum (forming local circuits within the neocortex. The claustrum forms long-distance connections with heterotopic cortical areas of the same and contralateral hemispheres. The neurons of the claustrum are mostly connected to heterotopic cortical areas, but there is some thalamic input. Innervation pathways from the claustrum are involved. in somatosensory, auditory, and visual functions.These parameters in this study indicated a high Rat ND Score (neurological deficit) in the normothermic group, which was also associated with the evaluation of the number of neurons in the claustrum undergoing apoptosis.

Claustrum provides long nerve pathways that connect somatosensory, auditory, visual pathways to conscious function⁴⁶, in which the damage to the claustrum area (in this study measured by the number of cells undergoing apoptosis), can be detected by examination of the Rat ND Score. The findings related to the most significant neurological deficits in the sensory components were also associated with the neuronal cells that make up the claustrum. Neurons in the claustrum receive convergent input from different cortical areas and send divergent outputs back to the corresponding cortical areas(Wang et al., 2017). Type I neurons make up about 85% of the claustrum area and are evenly distributed throughout. Type 1 neurons have spiny dendrites with axons projecting outward from the claustrum area. Type I neurons represent excitatory neurons that send projections to and receive projections from the cortex. The existence of these characteristics causes the claustrum to function as a detector, modulator, as well as an integrator of oscillations and synchronization of nerve functions⁴⁸.Based on these findings, it is very possible that there is a correlation between neuronal cell apoptosis in the claustrum (which indicates damage), which is associated with neurological deficits characterized by high ND Rat score parameters (sensory deficit, motor deficit, hearing, vision), reinforced by The results of the graphic analysis showed a linear correlation with the number of neurons undergoing apoptosis.

This study sheds information on prospective treatment strategies for neurological diseases by addressing the time required to produce hypothermia and the ensuing neurobehavioral consequences in experimental mice. However, several limitations warrant consideration. Firstly, the study's sample size and scope may limit the generalizability of its findings. Expanding the sample size and incorporating diverse experimental conditions could provide a more comprehensive understanding of the variables at play. Additionally, the study primarily focuses on neurobehavioral outcomes in mice, necessitating further research to extrapolate these findings to human neurological conditions. Incorporating translational research methodologies and clinical trials could bridge this gap and enhance the relevance of the study's findings to clinical practice.

Additionally, although the study found a strong link between claustrum neuron cell death and neurobehavioral impairments, the underlying mechanisms are still not yet fully understood. Subsequent research endeavours may focus on deciphering the biochemical processes implicated in this correlation, thereby potentially revealing innovative treatment targets for neurological conditions. Furthermore, investigating how genetic predispositions, environmental influences, and therapeutic interventions affect neurobehavioral outcomes may improve our knowledge of neurological pathology and guide the development of individualized treatment plans. The utilisation of sophisticated neuroimaging techniques and molecular biology methodologies in collaborative interdisciplinary research has the potential to facilitate the understanding of neurological illnesses and facilitate the development of tailored treatment therapies that will enhance patient outcomes.

CONCLUSION:

The HBN1 as a regulated hypothermia agent has been shown to be great potential in improving neurobehaviour function, after cardiac arrest. However, the hypothermia state in the force hypothermia group (using ice packs) was achieved faster when compared to the regulated hypothermia using a pharmacological approach (HBN-1).In this situation, HBN-1 provides a promising alternative. However, data regarding the use of HBN-1 is still scarce. Further research related to the use of HBN-1 in experimental animals with a larger number of samples and replication needs to be done. Additional aspect which need to be studied is the safety of using this pharmacological agent.

DECLARATION OF INTEREST:

Declared none.

ETHICS:

The study has been tested for ethical feasibility by the Research Ethics Commission of the Faculty of Veterinary Medicine, Airlangga University, Surabaya, the Animal Care and Use Committee (ACUC) No 2.KE.015.02.2020.

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Received on 13.11.2023 Modified on 09.02.2024

Research J. Pharm. and Tech 2024; 17(11):5632-5641.

DOI: 10.52711/0974-360X.2024.00858