INTRODUCTION:

Thyroid gland produces thyroid hormones such as T3 and T4 under the influence of Thyroid stimulating hormone (TSH) produced from anterior pituitary gland and are transported to various target tissue by binding with transporter proteins for various biological functions.

Hypothyroidism is a state of underactive thyroid when the thyroid gland makes insufficient thyroid hormones¹. Although the disease is more common among people aged 60 years, it is more pronounced among females and can contribute to clinical complications such as hypercholesterolemia, Myxedema, coma etc.,^1,2 Some of the common causes of hypothyroidism include Hashimoto’s disease, thyroiditis (inflammatory thyroid), congenital hypothyroidism (genetic defect by birth), treatment of hypothyroidism by radiation or by thyroidectomy, chronic inadequate or excessive intake of dietary iodine and pituitary and or hypothalamus dysfunction^1,3. Hashimoto’s disease is goitrous condition affecting over 5% of Indian population^4,5. It is an autoimmune disorder where the body’s immune system attacks its own cells of thyroid gland causing defective functions of thyroid gland^1,5. Vascular endothelial growth factor A (VEGF A) is essential for formation of new blood vessels and tissue repair after brain injury⁶ and has diagnostic roles in various thyroid and breast cancer disease^7,8. Chronic brain injury may pave for neurodegenerative disorders if left untreated⁹. Neurospecific enolase (NSE) and s100b are some of the promising biomarkers of brain injury and tumor marker ¹⁰. Neuron specific enolase (NSE) is localized in the neurons/ neuroendocrine cells and is elevated in the circulation on post-anoxic brain injury which may contribute to the development of thyroid dysfunction¹¹. In this study, we aim to assess and correlate the serum levels of VEGFA with the brain injury markers such as Neurospecific enolase (NSE) and s100b.

METHODS:

Ethical statement:

The study was carried out after getting the approval from the Institute Human ethics committee vide registered number MGMCRI/2023/02/IHEC/106 and the informed consent was obtained from all the study participants. All the procedures were carried out as per the ICMR, India 2017 guidelines and in accordance with Declaration of Helsinki of 1975 (revised 2013).

Study subjects:

The study subjects were recruited from the Department of Medicine, Mahatma Gandhi Medical College and Research institute, Puducherry, one of the premier tertiary care centers of the region. A total of 131 study subjects comprising 68 patients diagnosed with hypothyroid were recruited along with age and sex matched control subjects (n=63) who have no present or past history of thyroid disorders for this case control study. The sample size was calculated using the estimated values of VEGF-A using Open epi software, version 3.1¹². Study subjects with clinical complications such as cardiovascular events, any history of autoimmune disease other than related to thyroid disorders, malignancies, bronchial asthma, inflammatory disorders, pregnancy and lactation were excluded from the study.

Study procedure and assessment:

After obtaining an informed consent, the study subjects were collected 2ml of fasting venous blood and the serum was separated by centrifugation at 3500rpm for 10 min to store immediately at -40°C until analysis. The biochemical investigations such as serum glucose (fasting and post prandial), urea and creatinine were measured using the commercially available kits in fully automated clinical chemistry analyser (M/s Roche, Cobas c311, Germany). The thyroid profiles such as free T3 (fT3), free T4 (fT4) and Thyroid stimulating hormone (TSH) were assessed in Chemiluminescense assay (M/s Roche, Cobas e411, Germany). Glycated hemoglobin was estimated by ion-exchange high performance liquid chromatography method using D10 fully automated Analyser (M/s Bio-Rad Inc, USA). The serum levels were assessed for anti-thyroglobulin (Anti-TgAb) and anti Thyroperoxidase antibody (Anti-TPOAb) to look for autoimmune thyroid disorders by using commercially available ELISA kits (M/s Calbiotech, USA). The serum VEGF A (M/s Raybiotech, GA, USA), NSE and s100b (M/s MyBiosource Inc, CA, USA) levels were estimated using commercially available ELISA kits as per the manufacturer’s instructions.

Statistical analysis:

The values are expressed as mean±S.D or median (IQR) based on the normality of the parameters that was assessed using shapiro wilk’s test and Kolmogorov smirnov test. The statistical analysis such as ANOVA and Kruskalwallis tests were carried out to compare the means between the groups using SPSS v 19.0 software. Pearsons’s and Spearmann’s correlation was assessed to correlation between the markers and the P-value <0.05 was considered significant.

RESULTS:

In the present study, the mean age of the study subjects between the study groups showed no significant change. The serum thyroid profile shows significant decrease in fT3 and fT4 levels between the groups. The serum TSH levels show an increase in hypothyroid compared to control. There is no significant change observed in the serum urea, creatinine and diabetic profiles such as Fasting blood glucose, postprandial blood glucose and HbA1c (Table1). It was observed that out of 68 samples that was recruited for the study, 15 found positive for TgAb reflecting 22% as Hashimoto’s thyroiditis. The serum VEGF A markers shows a significant decrease in hypothyroidism compared to the control subjects. The brain injury markers show a significant decrease in hypothyroid study subjects compared to control subjects (Table 1). In order to assess the possible correlation between serum VEGF A and the brain injury markers, correlation analysis was carried and it was observed that the both the markers NSE and s100b showed a positive correlation with the serum VEGFA markers with correlation coefficient of 0.353 and 0.496 respectively (Table 2, Figure 1a,b).

ROC analysis was carried out to assess the diagnostic accuracy of the best cutoff values along with sensitivity, specificity and 95% CI of the serum VEGFA, NSE and s100b in predicting possible brain injury in hypothyroidism (Figure 2). The serum VEGF A had the best in predicting the hypothyroidism with possible brain injury at the cutoff value of 1254.5ng/mL showed AUC of 0.649 with 95% CI: .507 -0.709; P<0.05 (62% sensitivity and 61% specificity). Similarly the cutoff value for predicting autoimmune hashimotos disease had cutoff value of 1111.5 with 0.683 AUC, 72% specificity, 67% sensitivity with 95% CI: 0.508 – 0.858; P<0.05. As there were significant difference observed for the serum levels brain injury markers, NSE and s100b in control and hypothyroidism, ROC analysis was carried out to predict the diagnostic accuracy of the best cutoff values for serum NSE and s100b as brain injury marker among hypothyroidism. The serum NSE had the best cutoff value of 915.18ng/mL for predicting brain injury among hypothyroidism showed AUC of 0.677 with 95% CI: .528 -0.826; P<0.05 (77% sensitivity and 68% specificity) and for predicting hashimotos, the specificity and sensitivity were found to be 70% and 79% respectively as shown in Ttable 3. The serum value of s100b for predicting brain injury among hypothyroidism had the best cutoff value as 7.05 with 64% specific and 62% sensitivity and 95% CI: 0.534 – 0.807; P<0.05 (Table 3).

Table 1: The levels of serum biochemical markers in Hypothyroid subjects compared to healthy control.

Biochemical markers	Control (n-63)	Hypothyroid (n-68)
Biochemical markers	Mean ±SD/ Median (IQR)	Mean ±SD/ Median (IQR)
Age (years)	40.3 ± 9.8	39.9 ± 9.5
FT3 (ng/dl)	3.04 (2.69 - 3.45)	2.65 (2.115 - 3.03)*
FT4 (ng/dl)	1.26 (1.12 - 1.42)	1.01 (0.83 - 1.11)*
TSH (µIU/L)	2.48 (1.52 - 3.20)	8.21 (6.41 - 17.61)*
Fasting blood Glucose (mg/dl)	99.00 (90.50 - 144.50)	94.00 (85.75 - 115.25)
Post prandial blood Glucose (mg/dl)	149.00 (110.25 - 202.50)	133.00 (107.25 - 202.75)
HbA1C (g%)	5.90 (5.60 - 7.30)	5.85 (5.4 - 6.55)
Urea (mg/dL)	24.00 (16.00 - 29.00)	20 (15 -27)
Creatinine (mg/dl)	1.02 (0.78 - 1.15)	0.9 (0.75 - 1.2)
Anti-TgAb (n,%)	0 (0)	15 (22)
VEGFA (pg/ml)	1579.00 (830.55 - 2801.5)	992.7 (655.3 - 1579.75)*
NSE (ng/ml)	1294.64 (256.70 - 4947.92)	342.26 (238.10 - 892.85)*
S100b (ng/ml)	9.70 (2.18 - 48.87)	5.99 (1.52 - 12. 69)*

Note: Data are reported as mean±standard deviation (S.D) or Median (Inter Quartile Range) or n(%). Significant differences (P<0.05, independent samples student’s t test) are indicated as follows: * vs control group are statistically significant. (Significant differences between other groups are not indicated.)

Table 2. Correlation between VEGF A with brain injury markers NSE and s100b

	NSE		s100b
	Pearson’s coefficient (r)	p value	Pearson’s coefficient (r)	p value
VEGF A	0.353	0.001	0.496	0.0001

Table 3: Sensitivities, specificities and cutoff values of VEGFA, NSE and s100b in hypothyroidism.

Variable	Grouping	AUC	Specificity (%)	Sensitivity (%)	Best cut of value	Units	P value	95% CI
VEGF	Control vs Hypothyroidism	.649	62	61	1254.5	pg/ml	0.045	0.507- 0.709
VEGF	Control vs Hashimotos	.683	72	67	1111.5	pg/ml	0.048	0.508- 0.858
NSE	Control vs Hypothyroidism	.677	68	77	915.18	ng/ml	0.017	0.528 -0.826
NSE	Control vs Hashimotos	.704	70	79	915.18	ng/ml	0.034	0.534 -0.873
S100b	Control vs Hypothyroidism	.671	64	62	7.05	ng/ml	0.022	0.534 - 0.807

AUC: Area Under the Curve; VEGF: Vascular Endothelial Growth Factor A; NSE: Neuron Specific Enolase; S100B: S100 calcium-binding protein B. The cutoff values were determined based on the Youden index to optimize sensitivity and specificity. Statistically significant P values (<0.05) indicate discriminatory potential of the biomarkers between controls and hypothyroid/Hashimoto’s thyroiditis subjects. Units are expressed in pg/ml or ng/ml as appropriate. Confidence intervals (CI) are presented for AUC values.

Figure 1 a,b: Correlation between VEGF A with brain injury markers NSE and s100b. VEGFA; Vascular endothelial growth factor A, NSE: Neuron specific enolase, s100b: s100 calcium binding protein beta subunit. The statistical analysis for correlation was done by Pearson’s correlation and it has been observed that there is a positive correlation between VEGFA levels and a) s100b and b) NSE with correlation coefficient of 0.496 and 0.53 respectively. A pValue <0.05 was considered statistically significant.

Figure 2. Receiver operating characteristic (ROC) along with sensitivities, specificities, at the best cutoff of VEGFA, NSE and s100b in hypothyroidism. Vascular endothelial growth factor (VEGFA), Neuron specific enolase (NSE) and s100b

DISCUSSION:

Hypothyroidism is characterized by insufficient production of thyroid hormones and has significant impacts on brain function leading to neurological symptoms. Thyroid hormones are essential for normal brain development, neuronal functioning and metabolism, and their deficiency causes neuroendocrine dysfunctions leading to cognitive impairment, psychiatric symptoms, neuropathy, cerebellar dysfunction, depression, slow thinking, myopathy and sleep disturbances^13,14. A chronic and severe hypothyroidism can occasionally cause life threatening myxoedema coma, white matter abnormalities and demyelination, hypoxic injury and developmental issues as in congenital hypothyroidism. Hormone replacement therapy, levothyroxine (synthetic T4) is the primary treatment to restore normal thyroid function and alleviate neurological symptoms¹⁵.

Studies have shown that early treatment in congenital hypothyroidism prevents irreversible brain damage¹⁶. Cognitive rehabilitation and psychiatric support may be required for persistent deficits and in some instances, physical therapy can help address motor dysfunction. Addressing hypothyroidism promptly and adequately is crucial to minimize neurological complications and prevent permanent brain damage.

The VEGF A functions as angiogenic factors and hypoxia induces its expressions for neovascularisation ¹⁷. In our study, we have found that the levels of VEGFA are increased in hypothyroidism compared to control study subjects. In the recent past, it has been found that VEGF has neuroprotective effect also on many types of neural cells including astroglial cells. The therapeutic role of levothyroxine reduces hypoxic ischemia insult by inducing VEGF expression and angiogenesis^18–22. However, in our study, no significant correlation was observed between the thyroid hormone levels with that of the VEGF although the VEGF and thyroid hormone levels were significantly decreased in hypothyroid group compared to control. Studies have observed on the occurrence of microvascular and cardiac disorders among subclinical hypothyroid with diabetes complications²³. This shows that there might be other factor which could possibly influence on the expression of VEGFA protein in hypothyroidism.

Hypothyroidism is found to be associated with neurological manifestations that alter the structural and functional aspect of brain leading to altered levels of brain injury markers. Some of the brain injury markers are Neuron specific enolase and s100b which can be used as a biomarker to identify the severity of brain injuries such as traumatic brain injury (TBI), neurodegenerative conditions, stroke etc., In our study, it has been found that there is a significant decrease in the brain injury markers NSE and s100b just as that of the serum VEGF levels. Similar observations were found on the s100b and NSE levels in patients with dementia and Alzheimer’s disease respectively^24,25. Interestingly, we have also found a positive correlation of serum VEGFA with both the markers ie., the NSE and s100b. The decreased in VEGF besides the neurobiomarkers NSE and s100b in hypothyroidism potentially shows a state of chronic injury and impaired repair mechanism of the brain²⁶.

Astrocytes are glial cells that act as mediators of thyroid hormone metabolism in the brain²⁷. S100b is released by astrocytes that are essential for neuroprotective events such as clearing excess neurotransmitters, regulating blood-brain barriers and promoting synapse formation. Hypothyroidism is associated with impaired functions of astrocytes and mental retardation in animals and humans^28–30. Neuron-Specific Enolase (NSE) is an isoenzyme of the glycolytic enzyme enolase that is found predominantly in neuron cells and neuroendocrine cells. Studies reporting levothyroxine treatment on TBI model in rats and neuroblastoma cell line model shows evidence of increasing VEGF and NSE mRNA expressions¹⁵. On the other hand, Studies have reported on low levels of both VEGF, s100b and NSE in paediatric patients with convulsive status epilepticus (CSE)³¹. A systematic review on the effect of antipsychotic drugs on s100b reveals a decreased levels of s100b in 5 major psychiatric disorders³². Although studies found on induction of hypothyroidism on treatment with antipsychotic and antidepressant drugs, Levothyroxine treatment elicits neuroprotective effects improving cognitive deficit and memory beside oxidative stress in experimental rat model of Huntington’s disease^33,34,18,19. In our study, hypothyroid subjects were treated with levothyroxine and have increased TSH levels indicating that in subclinical hypothyroidism with high TSH level could be an inducer of neurological manifestation³⁵. Some studies have shown that natural adjuvants along with the conventional treatment may aid in better management of the complications^36–38. Despite of all these factors, the knowledge and awareness about the complications of thyroid disorders among patients is crucial for management of the disorder³⁹. Hence, in our study, although, we have found a decrease in VEGF that was positively correlated with brain injury markers NSE and s100b markers, the influence of thyroid hormone on these markers are inconclusive which needs to be addressed while managing hypothyroidism to prevent the neurodegenerative changes.

CONCLUSION:

A decrease in VEGF along with brain injury markers such as s100b and NSE were observed in hypothyroidism compared to control. Also a positive correlation was observed between the serum VEGF levels with that of the s100b and NSE marker levels. Assessment of NSE and s100b along with thyroid profile may be used as prognostic marker in preventing the onset of neurological manifestations in hypothyroidism.

LIMITATIONS OF THE STUDY:

The study was conducted with less sample size and the effect of levothyroxine might interfere with the assessment of brain injury marker in predicting the complex etiology of neurological manifestations. Also, the subgrouping of hypothyroid as hashimotos disease was based only on positive finding of TgAb and not by TPOAb.

CONFLICT OF INTEREST:

The Author do not have any conflict of interest.

ACKNOWLEDGEMENT/DECLARATION OF INTEREST:

The author thanks the host institute for providing facility to carry out this project.

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Received on 21.02.2025 Revised on 25.08.2025

Accepted on 31.12.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):1995-2000.

DOI: 10.52711/0974-360X.2026.00285

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