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| RESEARCH ARTICLE |
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| Year : 2023 | Volume
: 55
| Issue : 4 | Page : 216-222 |
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Role of vascular endothelial growth factor in tuberculous meningitis: A prospective study from a tertiary care center in North India
Devesh Kumar1, Deependra Singh2, Tanish Modi2, Riya Sharma2, Anand Keshav Awasthy2, Ritu Shree2, Manoj Kumar Goyal2, Dhawal Narang2, Siddharth Chand2, Sameer Vyas3, Kusum Sharma4, Navneet Sharma1, Rudresh Gowda1, Abeer Goel2, Rajeev Ranjan2, Manish Modi2
1 Department of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Neurology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Radiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
4 Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
| Date of Submission | 20-Oct-2022 |
| Date of Decision | 13-Jul-2023 |
| Date of Acceptance | 13-Jul-2023 |
| Date of Web Publication | 11-Sep-2023 |
Correspondence Address:
Rajeev Ranjan
Department of Neurology, Postgraduate Institute of Medical Education and Research, Chandigarh
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/ijp.ijp_743_22
INTRODUCTION: The status of vascular endothelial-derived growth factor (VEGF) in the pathogenesis of tuberculous meningitis (TBM) remains far from clear. We prospectively evaluated the role of serum and cerebrospinal fluid (CSF) VEGF in TBM.
PATIENTS AND METHODS: This prospective study was conducted at a tertiary care center in North India from January 2018 to June 2019. Consecutive drug-naive patients (n = 82) of TBM diagnosed on the basis of modified Ahuja's criteria were included in the study. The results were compared with 49 control subjects (n = 49). Serum and CSF VEGF were done in all the cases and controls. Follow-up serum VEGF levels were done in 34 patients after 3 months of completion of antitubercular therapy. The VEGF levels were estimated using the human VEGF enzyme-linked immunosorbent assay kit.
RESULTS: The mean age was 29.9 ± 13.1 years. The study group consisted of 33 (40.2%) men and 49 (59.8%) women. BACTEC MGIT960 was positive in 15 (18%) patients while multiplex tuberculosis polymerase chain reaction was positive in 73 (89%) patients. Levels of VEGF in serum and CSF of TBM patients were not elevated when compared to controls. There was no association between final outcome in TBM and decrease in serum levels of VEGF at follow-up.
CONCLUSION: VEGF may not be playing a significant role in the pathogenesis of TBM. Future studies with larger sample size may clarify the status of VEGF further in TBM.
Keywords: Antitubercular therapy, prognosis, tuberculous meningitis, VEGF
How to cite this article:
Kumar D, Singh D, Modi T, Sharma R, Awasthy AK, Shree R, Goyal MK, Narang D, Chand S, Vyas S, Sharma K, Sharma N, Gowda R, Goel A, Ranjan R, Modi M. Role of vascular endothelial growth factor in tuberculous meningitis: A prospective study from a tertiary care center in North India. Indian J Pharmacol 2023;55:216-22 |
How to cite this URL:
Kumar D, Singh D, Modi T, Sharma R, Awasthy AK, Shree R, Goyal MK, Narang D, Chand S, Vyas S, Sharma K, Sharma N, Gowda R, Goel A, Ranjan R, Modi M. Role of vascular endothelial growth factor in tuberculous meningitis: A prospective study from a tertiary care center in North India. Indian J Pharmacol [serial online] 2023 [cited 2023 Sep 23];55:216-22. Available from: https://www.ijp-online.com/text.asp?2023/55/4/216/385500 |
| » Introduction | |  |
Despite decades of research, the outcome of tuberculous meningitis (TBM) remains poor with death in 25% and significant residual disability in approximately one-third of survivors.[1],[2],[3]
The immune response of host to infection with tubercular bacilli can both be good or bad depending on its severity. In fact, the response of host immune system to tubercular bacilli contributes to death as well as other complications in TBM.[4] This is well exemplified by the routine use of steroids along with antitubercular treatment (ATT) in TBM to suppress the immune response to tubercular bacilli.[5]
A major cause of various complications in TBM is brain infarction which affects approximately 40% of patients.[6] The most important reason for the occurrence of brain infracts is outpouring of thick exudates at the base of the brain which leads to strangulation and occlusion of intracranial vessels. Accordingly, various cytokines, chemokines, and VEGF have been implicated in the pathogenesis of cerebral infarctions in TBM.[5],[6],[7],[8]
Vascular endothelial-derived growth factor (VEGF), also called vascular permeability factor, is a 36–46 kDa homodimeric glycoprotein which regulates permeability of vascular endothelium. It contributes to the pathogenesis of TBM by damaging blood–brain barrier (BBB) with attendant cerebral inflammation and edema. VEGF does play a role in damaging BBB in bacterial meningitis, central nervous system (CNS) neoplasms, and brain infarctions.[6] However, only a handful of studies[9],[10],[11] have evaluated the role of VEGF in TBM. Even these suffered from limitations of small sample size and use of computed tomographic scan rather than magnetic resonance imaging (MRI) to detect brain infarcts. In the absence of clear data from well-conducted prospective studies, the impact of VEGF in the pathogenesis of TBM is yet not clear. If VEGF is found to play a major pathogenic role in CNS TB, future treatment protocols based on anti-VEGF therapies may help in improving outcome in TBM. Thus, we prospectively evaluated the role of VEGF in TBM.
Aims and objectives
The aims and objectives of the study were to determine the role of VEGF in the pathogenesis of TBM.
| » Patients and Methods | | |
The present prospective study was done at a university standard hospital from January 2018 to June 2019. Diagnosis of TBM was made on the basis of the modified Ahuja's criteria.[1],[2] We included 82 consecutive newly diagnosed patients of TBM who had not received ATT before the collection of samples. The results were compared to healthy controls (n = 49) consisting of patients who underwent cerebrospinal fluid (CSF) examination for diagnostic purposes and during surgery under spinal anesthesia. Control subjects did not have any active CNS disease or any other disease which is known to elevate VEGF levels. The study was approved by the Ethics Committee of the institute wide order number NK/4247/MD/2666-67. Patients and controls were included and excluded based on the following criteria.
Inclusion criteria
- Age more than 14 years
- Patients being diagnosed as TBM using modified Ahuja's criteria.
Exclusion criteria
- Pregnancy
- Presence of organisms other than Mycobacterium tuberculosis
- Patients on antitubercular therapy or other forms of immunosuppressive therapy
- Presence of disorders (other than TBM) which may elevate VEGF levels.
History was obtained and meticulous systemic and neurological examinations were performed on all patients. All the details were noted on a predesigned pro forma. All patients underwent detailed hemogram and biochemical investigations including (but not limited to) hemoglobin; total and differential leukocyte counts; erythrocyte sedimentation rate; platelet counts; blood sugars; renal, liver, and thyroid function tests; serum electrolytes; and serum calcium and phosphorus. The CSF was EXAMINED for cells, protein, glucose, acid-fast bacillus (AFB) smear, adenosine deaminase, multiplex polymerase chain reaction (PCR) for mycobacterial DNA, GeneXpert for MTB, culture and drug susceptibility testing for MTB on Lowenstein–Jensen medium, BACTEC Automated Blood Culture Systems, cultures for bacteria and fungi, and antigen testing for Cryptococcusetc . MRI brain with contrast was done in all patients at baseline. All patients received four drugs ATT (antituberculous treatment) namely rifampicin, isoniazid, pyrazinamide, streptomycin (RHZS) with steroids. Ventriculoperitoneal shunting was done wherever needed. The cases were reassessed by clinical examination every monthly and radiological examination after 3 months of treatment. They also underwent neuroimaging and clinical evaluation on an as-and-when-required basis. Eventual outcome was determined through the Glasgow Outcome Scale[12] and Schwab and England Activities of Daily Living (S and E ADL) Scale.[13] The British Medical Research Council criteria were used to determine the grade of TBM.[14] The authors obtained written informed consent from all the participants or their relatives (if the primary subject was <18 years of age or had obtunded sensorium).
Five milliliters of blood and 7–8 mL of CSF were collected with full aseptic precautions from all patients at baseline for the estimation of VEGF levels. After 3 months of treatment with ATT, blood samples were recollected for VEGF estimation. Two milliliters of CSF was taken from controls during lumbar puncture carried out for anesthetic purposes during surgery after taking consent. Serum was obtained from blood and taken in an autoclaved tube. It was then centrifuged at 3000 revolutions/min for 10 min and then kept at a temperature of −80°C.
The VEGF was estimated by human VEGF DIACLONE enzyme-linked immunosorbent assay kit, a solid-phase kit designed to determine VEGF level.
Statistical analysis
All data were analyzed using the IBM Statistical Package for the Social Sciences (SPSS) for Windows, version 25.0. Armonk, NY: IBM Corp. Demographic data were analyzed using mean, median, and range. To analyze discrete variables, the Chi-square test was utilized, whereas the Mann–Whitney test was used to analyze continuous variables. Statistical significance was defined as P ≤ 0.05. The Student's t-test was used to compare VEGF levels at baseline and 3 months after ATT.
| » Results | | |
Demographic and clinical profile
The mean age of TBM patients (29.9 ± 13.1 years) was significantly less (P < 0.01) than control population (41.8 ± 9.5 years). There were 33 (40.2%) men and 49 (59.8%) women in the TBM group and 22 (44.9%) men and 27 women (55.1%) in the control group. The male-to-female ratio was comparable among patients and controls. These as well as rest of the demographic and clinical data are summarized in [Table 1]. | Table 1: Clinical and demographic profile of patients with tuberculous meningitis
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Radiological and cerebrospinal fluid data
Baseline MRI (n = 77) was abnormal in 76 (98.7%) patients. The most common MRI abnormality included the presence of exudates seen in 85.7% of patients followed by hydrocephalus in 62.2% of patients. Evidence of brain infarction was seen in 24.4%. CSF was abnormal in 76/82 (92.7%) of patients. The remaining six patients had unmistakable evidence of CNS tuberculosis on neuroimaging. AFB culture (BACTEC MGIT960) was positive in 15 (18%) patients while multiplex PCR for tubercular bacillus was positive in 73 (89%) patients. The MRI and CSF results are shown in [Table 2]. | Table 2: Cerebrospinal fluid and radiological data of patients with tuberculous meningitis
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Final outcome among patients with tuberculous meningitis
The final outcome in TBM was defined either by:
- Death (n = 24) or alive (n = 58) or
- Using the Glasgow Outcome Scale score (good – 5, moderate – 4, and poor – 1–3) or Schwab and England ADL Scale score (100%–80% – good, 60%–70% – moderate, and <60% – poor). The outcome was taken as poor if there was death or if any scale revealed poor outcome, moderate if any scale revealed moderate outcome, and good if both scales have good outcome.
Overall, death occurred in 24 (29.3%) patients. Forty (48.8%), 12 (14.6%), and 30 (36.6%) patients had good, moderate, and poor outcomes, respectively.
Determinants of poor outcome
We further analyzed as to if various clinical, biochemical, CSF, or radiological parameters [Table 3] can predict the final outcome in TBM. The following associations were found as follows: | Table 3: Correlation of clinical and radiological parameters with final outcome in tuberculous meningitis
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- Positive association between low Glasgow Coma Scale and death as well as poor final outcome
- Trends between TBM Stage III and death (P = 0.08) and poor outcome without death on any of the scales compared to lower clinical stage of TBM
- Trend (P = 0.08) between altered sensorium and poor outcome as defined by either of the two scales
- A trend toward death and the presence of exudates (P = 0.06) and cerebral edema (P = 0.09)
- Positive association between poor outcome (on either of the scales) and the presence of cerebral infarcts (P = 0.02)
- Positive association between the presence of cerebral tuberculomas and good outcome (determined by the presence or absence of mortality) (P = 0.02) and a trend between the presence of cerebral tuberculomas and good outcome as defined by either of the two scales (P = 0.06).
VEGF in serum and cerebrospinal fluid samples of tuberculous meningitis and controls
Comparison of serum and cerebrospinal fluid VEGF in cases versus controls
In the present study, we evaluated the role of VEGF in the pathogenesis of TBM. The levels of serum and CSF VEGF were measured in TBM and compared with controls. On analysis, serum and CSF VEGF were greater (though statistically insignificant; P = 0.01) in TBM than controls [Table 4]. | Table 4: Serum and cerebrospinal fluid vascular endothelial-derived growth factor in patients with tuberculous meningitis versus controls
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Correlation between VEGF and various clinical parameters
We also compared serum and CSF VEGF with the stage of TBM and with the final outcome among patients with TBM. On analysis, there was no correlation of the severity of TBM (as suggested by clinical staging) or final outcome of TBM with either serum or CSF VEGF.
Correlation between VEGF and various radiological parameters
We determined the role of VEGF in radiological compilations of TBM such as hydrocephalus, exudates, infarcts, tuberculomas, border zone encephalitis, and cerebral edema. Analysis revealed a positive association (P = 0.01) between serum VEGF and exudates on MRI. There was no correlation between serum and CSF VEGF with any other radiological parameter.
Follow-up serum VEGF levels
In this present study, we also determined levels of serum VEGF at follow-up. Follow-up VEGF testing was done in 34 patients. At follow-up serum VEGF decreased only in 15 (44.1%) patients. When compared, change in serum levels of VEGF did not have any bearing on the final outcome. These results are shown in [Table 5]. | Table 5: Follow-up serum vascular endothelial-derived growth factor levels and their influence on the outcome as defined by dead or alive
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| » Discussion | | |
VEGF family consists of VEGF A, B, C, D, E, and placental growth factors. These act through tyrosine kinase receptors. In general, when we talk of VEGF, we refer to VEGF A, a 45 kDa glycoprotein produced by many different cell types. Primarily released from vascular endothelial cells in response to hypoxia, VEGF potently stimulates angiogenesis. In cerebral ischemia, VEGF can have both pathogenic and protective roles depending on the stage of disease process. While, in early stages, VEGF contributes to brain damage through increase in vascular permeability with consequent cerebral inflammation and edema; in the late stages, it protects against ischemia through the formation of new blood vessels. As disruption of BBB (with increased vascular permeability and influx of inflammatory cells) remains a major factor for cerebral damage in TBM, it is likely that VEGF contributes to the pathology of TBM at least in the early stages of disease process. VEGF also stimulates the genesis of nitric oxide resulting in vasodilatation with increased blood flow and consequent cerebral vasodilatation. Accordingly, it is imperative to study the role of VEGF in TBM where vascular complications are important determinants of death and other disabling sequelae.[9],[10],[11]
Matsuyama et al.[9] found a significant increase in VEGF (in blood and CSF) in TBM (n = 28) than other CNS infections (n = 31). Follow-up VEGF levels decreased in patients who showed clinical improvement (n = 12). They stressed on the importance of VEGF in TBM. In another study conducted on pediatric population,[10] CSF VEGF was significantly higher in TBM (n = 26) than healthy children (n = 20). There was a positive correlation between CSF mononuclear cell count and VEGF levels. Thus, inflammatory cells in CSF secrete VEGF which, in turn, damages BBB. The authors suggested that steroids may exert their benefit in TBM by antagonizing the effects of VEGF. Husain et al.[11] reported significantly higher blood and CSF VEGF in ongoing (n = 20) compared to inactive TBM (n = 20).
The demographic, clinical, laboratory, positron emission tomography, and MRI data of the present study were consistent with that reported previously from our center.[1],[2],[15] Regarding serum and CSF VEGF, our results contrasted with previous studies as we did not find a role of VEGF in the pathogenesis of TBM. Although blood and CSF VEGF were more in TBM than healthy subjects, the difference did not reach statistical significance. Our results were similar to those of Misra et al.[6] who reported insignificantly higher serum VEGF in TBM (n = 40) patients compared to controls. Similar to Misra et al.,[6] the present study did not find any correlation between VEGF levels and disease severity or time of presentation.
Among various radiological parameters, we did find a significant correlation between basal exudates and serum VEGF levels but not with CSF VEGF levels. The association between increased serum VEGF and basal exudates can be explained due to VEGF-induced vasodilatation and increased vascular permeability. However, the above inference is putative as there was no association between CSF VEGF levels and the presence of basal exudates. Similar to the study by Misra et al.,[6] we did not find any effect of correlation between the presence or absence of cerebral infarction and VEGF levels in TBM.
In our study, we compared change in the value of VEGF after treatment with ATT for 3 months. Unlike a previous study,[9] we did not find any correlation between decrease in levels of VEGF with the final outcome.
Our findings are significant. Our results could not reiterate the previously suggested role played by elevated VEGF in the pathogenesis of TBM. The likely reason could be the unique genetic structure of our population wherein a different set of cytokines rather than VEGF may be more operative in the pathogenesis of TBM. The point in favor of the above hypothesis is the fact that a previous study from Indian subcontinent also did not find a significant role of VEGF in TBM.
| » Conclusion | | |
To conclude, out study did not find a signifcant role of VEGF in pathogenesis of TBM.
Major positive points of our study were big sample size and the fact that MRI brain was carried out in all patients both at baseline and at follow-up. Another major strength of the present study is that 89% of our patients fell within the category of definite TBM. Future studies with larger sample size may help in clearing the role of VEGF further in TBM.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
| » References | | |
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| 11. | Husain N, Awasthi S, Haris M, Gupta RK, Husain M. Vascular endothelial growth factor as a marker of disease activity in neurotuberculosis. J Infect 2008;56:114-9. |
| 12. | Jennett B, Bond M. Assessment of outcome after severe brain damage. Lancet 1975;1:480-4. |
| 13. | Ramaker C, Marinus J, Stiggelbout AM, Van Hilten BJ. Systematic evaluation of rating scales for impairment and disability in Parkinson's disease. Mov Disord 2002;17:867-76. |
| 14. | Lamba PA, Bhalla JS, Mullick DN. Ocular manifestations of tubercular meningitis: A clinico-biochemical study. J Pediatr Ophthalmol Strabismus 1986;23:123-5. |
| 15. | Jain A, Goyal MK, Mittal BR, Sood A, Singh H, Vyas S, et al. 18FDG-PET is sensitive tool for detection of extracranial tuberculous foci in central nervous system tuberculosis – Preliminary observations from a tertiary care center in Northern India. J Neurol Sci 2020;409:116585. |
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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