INTRODUCTION

Neonatal hypoxic-ischemic encephalopathy (HIE) [1,2], develops when the neonatal brain is subjected to insufficient oxygenation and impaired cerebral blood flow during late gestation, labor, delivery, or the early postnatal phase. Its clinical manifestations are highly variable, encompassing mild signs such as irritability and transient hypotonia to severe forms of encephalopathy characterized by frequent seizures, marked alterations in consciousness, and profound deficits in motor function [3]. The pathophysiology of HIE is driven by a complex, multi-stage cascade of cellular and molecular disturbances initiated immediately after the hypoxic-ischemic insult [4]. This biphasic nature of injury provides a critical therapeutic window for neuroprotective interventions.

The long-term consequences of HIE are profound, with survivors frequently experiencing cerebral palsy, intellectual disability, epilepsy, and sensory impairments that significantly impact quality of life and impose substantial socioeconomic burdens on families and healthcare systems [5]. Currently, therapeutic hypothermia remains the only established neuroprotective treatment for moderate to severe HIE in term and late preterm infants [6]. However, despite its proven efficacy, therapeutic hypothermia provides only modest neuroprotection, with approximately 45–50% of treated infants still experiencing death or major disability, highlighting the critical need for adjunctive or alternative therapeutic strategies [7]. Furthermore, hypothermia primarily targets the secondary phase of injury through metabolic suppression but does not directly address the underlying molecular mechanisms of neuronal death, including oxidative stress, inflammation, and excitotoxicity that contribute to ongoing brain damage [8,9].

Recent clinical trials investigating adjunctive therapies such as erythropoietin, melatonin, and xenon have shown mixed results, with some promising preclinical findings not translating to significant clinical benefits [10–13].

The primary phase is characterized by immediate cell death through necrosis, particularly in regions with high metabolic demand such as the basal ganglia, thalamus, and perirolandic cortex [14,15]. The secondary injury phase is particularly relevant to oxidative stress mechanisms, as reperfusion paradoxically increases reactive oxygen species generation while cellular antioxidant defenses remain compromised [15,16].

Reactive oxygen species play a central role in the pathogenesis of hypoxic-ischemic brain injury, with their generation occurring through multiple pathways during both the primary and secondary phases of injury [17,18]. The activation of enzymes such as xanthine oxidase, NADPH oxidase, and cyclo-oxygenase contributes to ROS production during the reperfusion phase [18,19].

ROS-mediated damage extends beyond lipid peroxidation to include protein oxidation and DNA damage. A third phase may occur days later as inflammatory cells infiltrate damaged brain regions and activated microglia generate additional ROS through NADPH oxidase activation. This prolonged oxidative stress contributes to the expansion of brain injury beyond the initial areas of damage and provides multiple opportunities for therapeutic intervention [18,20–22].

Vitamin E and CoQ₁₀ have emerged as particularly promising candidates due to their excellent safety profiles, favorable pharmacokinetic properties, and demonstrated neuroprotective effects in experimental models of HIE. Vitamin E, a lipophilic antioxidant that protects cellular membranes from lipid peroxidation, and CoQ₁₀, which serves dual roles as a mitochondrial electron transport chain component and potent antioxidant, represent mechanistically distinct yet complementary approaches to combating oxidative stress in neonatal HIE [23–25]. Earlier, simultaneous administration of metformin and CoQ₁₀ has been reported to enhance anti-inflammatory, antidiabetic action [26–28], indicating CoQ₁₀ contributes to metformin action.

Data on their combined effects on neonatal hypoxic-ischemic encephalopathy injury are lacking. Therefore, the aim of the present study is to evaluate the effects of metformin, vitamin E and CoQ₁₀ in neonatal rat model of hypoxic-ischemic encephalopathy and associated cognitive complications.

MATERIALS AND METHODS

Metformin was obtained as a gift sample from Emcure Pharmaceutical, Pune, Maharashtra, whereas CoQ₁₀ was purchased by SV Agro Mumbai, Maharashtra. Vitamin E was procured from Matrix Life Science Private Limited (Aurangabad, Maharashtra). All other chemicals and reagents used in this work were of analytical grade.

INDUCTION OF HIE

Induction of hypoxic-ischemic encephalopathy [HIE] was a induced in rat pups via unilateral carotid artery ligation followed by a low oxygen environment on PND 7 (Table 1).

Briefly, pups were anesthetized with 5% inhaled isoflurane followed by 2% for maintenance. The right common carotid artery was carefully isolated from the vagus nerve and ligated. The wound was closed with surgical glue and the pups were allowed to recover in a cold-water bath with a set temperature of 37°C for 20 min and rectal temperature was recorded throughout the duration. Pups were then placed in a hypoxic chamber (92% N, 8% O₂) for 120 min [29].

During the hypoxia procedure, surface body temperature was controlled using cold water bath with a set temperature of 37°C and rectal temperature was recorded throughout the time. Subsequently, pups were allowed to recover for 20 min followed by administration of vehicle (0.9% saline) or vitamin E or metformin or CoQ_10, after which pups were kept at room temperature (Table 2). Euthanasia on PND 14, pups were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) via ip injection and then decapitated. The brain of each pup were dissected out, weighed, washed with chilled saline, and stored at 80°C for oxidative stress markers.

RESULTS

BIOCHEMICAL ASSAYS

Effect of Treatment Drug on Oxidative Stress Markers

Challenging the animals with HIE followed by treatment with hypothermia significantly caused oxidative damage (Table 4) as indicated by increased in lipid peroxidation (MDA level), it caused by oxidative damage significant in brain cortex of HYPO group while decreased superoxide dismutase (SOD), and glutathione (GSH) activity in whole brain region. Pre-treatment with MET (200 mg/kg), Vit E (100 mg/kg) and CoQ₁₀ (200 mg/kg) significantly restored depleted GSH (glutathione) and catalase activity as well as attenuated elevated lipid peroxidation of the brain, as compared to their respective control and HYPO group (Figures 1–3).

Table 4 - Effect of treatment on oxidative stress markers

Groups	SOD (IU/mg TP)	GSH (µg/mg TP)	LPO (µM/mg TP)
SHAM	13.04 ± 0.32	83.20 ± 1.49	6.16 ± 0.47
HIE + Hypothermia [HYPO]	3.71 ± 0.07 a	11.05 ± 0.48 a	15.24 ± 0.61 a
HIE + Hypothermia + MET + Vit E	9.10 ± 0.60	24.68 ± 2.91 a	8.76 ± 0.29 a b
HIE + Hypothermia + MET + CoQ10	9.07 ± 2.40	21.96 ± 4.02 a	8.15 ± 0.07 b
HIE + Hypothermia + MET + Vit E + CoQ10	11.56 ± 0.88 b	69.73 ± 5.06bcd	8.70 ± 0.58 a b

All data were expressed as Mean ± SEM. Statistical difference were determined by One-way ANOVA followed be Bonferroni’s Multiple Comparison test. Where the values are expressed as Mean ± SEM (n = 4). Where^ap < 0.05 vs SHAM,^bp < 0.05 vs HYPO,^cp < 0.05 vs MET + Vit E and^dp < 0.05 vs MET + Vit E + CoQ₁₀.

Figure 1 - Graph depicting SOD level in the brain. All data were expressed as Mean ± SEM. Statistical difference were determined by One-way ANOVA followed be Bonferroni’s Multiple Comparison test. Where the values are expressed as Mean ± SEM (n = 4). Where ^ap < 0.05 vs SHAM, ^bp < 0.05 vs HYPO, ^cp < 0.05 vs MET + Vit E and ^dp < 0.05 vs MET + Vit E + CoQ₁₀.

Figure 2 - Graph depicting GSH level in the brain. All data were expressed as Mean ± SEM. Statistical difference were determined by One-way ANOVA followed be Bonferroni’s Multiple Comparison test. Where the values are expressed as Mean ± SEM (n = 4). Where ^ap < 0.05 vs SHAM, ^bp < 0.05 vs HYPO, ^cp < 0.05 vs MET + Vit E and ^dp < 0.05 vs MET + Vit E + CoQ₁₀.

Figure 3 - Graph depicting LPO level in the brain. All data were expressed as Mean ± SEM. Statistical difference were determined by One-way ANOVA followed be Bonferroni’s Multiple Comparison test. Where the values are expressed as Mean ± SEM (n = 4). Where ^ap < 0.05 vs SHAM, ^bp < 0.05 vs HYPO, ^cp < 0.05 vs MET + Vit E and ^dp < 0.05 vs MET + Vit E + CoQ₁₀.

TTC Staining for Infarct Volume

Ischemia injury causes infraction in the brain regions. In the HYPO group show severe cerebral infarction due to extensive damage inflicted by HYPO injury as compared with SHAM group. Pre-treatment with MET, Vit E and CoQ₁₀ showed significant reduction in infarct area as compared with HYPO group. Whereas pre-treatment with MET + Vit E + CoQ₁₀ along with hypothermia showed significant reduction in infarct area as compared with HYPO and MET, Vit E and CoQ₁₀ treated group (Table 6, Figure 4).

Table 6 - Effect of treatment drug on infarct volume

Groups	% Brain infarct area
SHAM	6.11 ± 1.36
HIE + Hypothermia [HYPO]	44.11 ± 0.76 a
HIE + Hypothermia + Met + Vit E	25.07 ± 1.37 a b
HIE + Hypothermia + Met + CoQ10	11.00 ± 0.63abc
HIE + Hypothermia + Met + Vit E + CoQ10	5.64 ± 0.21bcd

All data were expressed as Mean ± SEM. Statistical difference were determined by One-way ANOVA followed be Bonferroni’s Multiple Comparison test. The values are expressed as Mean ± SEM (n = 4). Where^ap < 0.05 vs SHAM,^bp < 0.05 vs HYPO,^cp < 0.05 vs MET + Vit E and^dp < 0.05 vs MET + Vit E + CoQ₁₀.

Figure 4 - Graph depicting % brain infarct area All data were expressed as Mean ± SEM. Statistical difference was determined by One-way ANOVA followed be Bonferroni’s Multiple Comparison test. The values are expressed as Mean ± SEM (n = 4). Where ^ap < 0.05 vs SHAM, ^bp < 0.05 vs HYPO, ^cp < 0.05 vs HYPO + MET + Vit E and ^dp < 0.05 vs HYPO + MET + CoQ₁₀.

Infarct analysis (Coronal brain sections 2 mm), stained with 2% TTC, showing stained brain slices infarct area after 15 min bilateral common carotid artery occlusions followed by 24 h reperfusion in SHAM, HYPO and treatment groups pups (Figure 5). The red area represents the non-ischemic (viable tissue) area (Figure 6), whereas the white area indicates ischemic area (non-viable tissue) in the coronal section.

Figure 5 - Effect of MET, Vit E and CoQ₁₀ on brain infarction Where A: SHAM; B: HYPO; C: HYPO + MET + Vit E; D: HYPO + MET + CoQ₁₀; E: HYPO + MET + Vit E + CoQ₁₀.

Figure 6 - Cerebral infarction. A: SHAM; B: HYPO; C: HYPO + MET + Vit E; D: HYPO + MET + CoQ ₁₀; E: HYPO + MET + Vit E + CoQ₁₀.

Figure 7 - Histopathology of brain.

DISCUSSION

The developing neonatal brain exhibits heightened susceptibility to oxidative stress due to several unique developmental characteristics that distinguish it from the mature brain. The high rate of brain growth during the perinatal period is associated with elevated oxygen consumption and metabolic activity, creating an environment prone to oxidative stress when oxygen delivery is compromised. Additionally, the process of myelination, which is actively occurring in the neonatal brain, involves the synthesis of lipid-rich myelin sheaths that are particularly vulnerable to lipid peroxidation [11]. This study demonstrates that metformin, vitamin E, and CoQ₁₀ offer significant neuroprotection against neonatal HIE. The triple therapy showed synergistic benefits by targeting mitochondrial dysfunction, oxidative stress, neuroinflammation, and BBB breakdown. The combined antioxidant and mitochondrial support provided by these agents appears to be the key mechanism.

Metformin further augmented neuroprotection by ameliorating mitochondrial dysfunction, reducing inflammatory signaling, and enhancing cellular resilience against metabolic collapse. However, the most profound protective effects emerged from the triple-combination regimen, which exhibited synergistic restoration of antioxidant defenses (SOD, GSH), marked suppression of lipid peroxidation (LPO), and normalization of BBB integrity as evidenced by reduced Evans blue extravasation [38].

The treatment groups demonstrated significant reductions in infarct volume on TTC staining, accompanied by preservation of pyramidal neuron density within the CA1–CA3 hippocampal regions, areas critically vulnerable to hypoxic-ischemic injury. Histopathological analyses confirmed reduced neuronal pyknosis, diminished cytoplasmic vacuolation, and attenuation of inflammatory cell infiltration in combination-treated animals, indicating effective mitigation of both necrotic and apoptotic pathways. Behavioral assessments including neurological deficit scoring, Y-maze performance and NORT outcomes further corroborated that antioxidant therapy yielded functional improvements consistent with enhanced neuronal survival and synaptic integrity [39].

Collectively, the results underscore that neonatal HIE is driven by a convergent cascade involving oxidative stress, mitochondrial impairment, excitotoxicity, neuroinflammation, BBB disruption, and secondary energy failure [40,41]. The superior efficacy of metformin + vitamin E + CoQ₁₀ suggests that targeting multiple pathophysiological checkpoints simultaneously is substantially more effective than monotherapy approaches. The observed synergistic interaction supports the hypothesis that combination antioxidant therapy can broaden the therapeutic window, enhance mitochondrial resilience, stabilize membrane structures and better preserve neurovascular unit function.

CONCLUSION

The ultimate goal of providing comprehensive neuroprotective therapy that combines the proven benefits of therapeutic hypothermia with the complementary mechanisms of antioxidant protection represents a realistic and achievable advance in neonatal care. The evidence presented in this research strongly supports the continued development of vitamin E and CoQ₁₀, both individually and in combination, as promising therapeutic strategies for one of the most devastating conditions affecting newborn infants. Further studies are required to evaluate the long-term treatment effect of this combination & estimation of cognitive biomarkers would be interesting. In conclusion, the triple therapy of metformin, vitamin E, and CoQ₁₀ significantly mitigates hypoxic-ischemic brain injury by targeting key mechanisms underpinning oxidative, metabolic, and inflammatory damage. The results support the emerging paradigm that combination antioxidant therapy represents a promising, mechanistically rational, and clinically relevant strategy for improving neurological outcomes in neonatal HIE.