Aneurysmal Subarachnoid Hemorrhage: Impact on Phenytoin Permeability across the Blood–Brain Barrier
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.288987
Source of Support: None, Conflict of Interest: None
Keywords: Aneurysmal subarachnoid hemorrhage, antiepileptic, aSAH, brain tumor, human brain, LC-MS/MS, phenytoin
Neha Dhir and Savita Verma Attri have equal contribution.
Subarachnoid hemorrhage (SAH) is a devastating neurological emergency that leads to significant mortality.,,, The rupture of the intracranial arterial aneurysm is the most common cause for nontraumatic subarachnoid bleed. Seizures are well-recognized complications following aneurysmal subarachnoid hemorrhage (aSAH), occurring in 20% of aSAH-affected patients especially within the first 24 hours of ictus.,,,, Seizures result in increased intracranial pressure causing hemodynamic instability, increased metabolic demand, and diminished oxygen supply precipitating to delayed neuronal injury., Thus, the use of antiepileptic drugs (AEDs) as prophylaxis in these patients is a common neurosurgical practice, especially when transcranial route is used for management.,,,, Various AEDs have been tried in such patients that include valproate, phenytoin (PHT), phenobarbital, carbamazepine, zonisamide, levetiracetam, and topiramate.,,, Among these AEDs, PHT is the frequently prescribed AED followed by phenobarbital and carbamazepine. However, the clinical practice of use of PHT in aSAH falls in a customary or narrative-based fashion, rather than evidence based., Emerging evidence also suggests that, use of PHT is associated with worsening of both neurologic and functional recovery in these patients.,,
Experimental and clinical studies have revealed that in aSAH there is profound damage to both capillary basement membrane and endothelial cells of blood–brain barrier (BBB). This is followed by the BBB disruption that leads to changes in its permeability, affecting the penetration of drugs and other substances across this barrier , which (the penetration of drugs), may be decreased, increased, or even unchanged.,,,,, In the available scientific literature, there is no corroboration to suggest the penetration of PHT through BBB in aSAH patients. Thus, the current study aimed to evaluate the alteration in PHT permeability across the BBB in aSAH patients as compared to those with brain tumors (no evidence of BBB permeability breach), which are selected as control.
A total number of 20 subjects (control = 10, cases = 10) were enrolled into the study from the Department of Neurosurgery at a tertiary care Hospital. The inclusion–exclusion criteria and demographic details for both aSAH (grade 3 or 4) and control groups are described in [Table 1] and [Table 2], respectively. In cases of anterior communicating artery ( ACoM) aneurysms (n = 5), gyrus resection was required and the brain tissue was subjected to PHT estimation. In cases of middle cerebral artery (MCA) and posterior communicating artery (PCoM) aneurysms, only those cases that warranted temporal lobe hematoma evacuation along with aneurysm clipping were included. For internal carotid artery bifurcation (ICA (b)) aneurysms, both patients had frontal lobe hematomas requiring evacuation along with aneurysm clipping. The brain tissue taken en-route for hematoma evacuation was collected for PHT estimation. Before enrollment, the purpose and nature of the study was explained to all the subjects. The willingness to participate was documented as written informed consent. The study was initiated after getting approval from the institutional ethics committee (NK/1791/Study/2173).
Sample collection and PHT estimation in the brain, plasma, and CSF
Both cases and controls, were administered a loading dose of PHT (20 mg/kg), infused in 5% dextrose at a rate, not more than 50 mg/min, and followed by a maintenance dose of 5 mg/kg. During surgical procedure, simultaneous samples of blood, CSF, and brain tissue were harvested for PHT estimation. Samples were stored at −80°C until analysis. For PHT quantification, respective samples were processed and PHT extraction was done using MassTox® TDM BASIC kit-A (Chromsystems Instruments and Chemicals GmbH, Germany; Order no. 92111/200, a commercially available kit) as per the manufacturer's instructions. QTRAP® 4500 series liquid chromatography-mass spectrometry (LC-MS/MS) system (AB Sciex Pvt. Ltd, USA) was used for PHT estimation in all three biological matrices. The entire system was connected to a computer where all data were collected and analyzed using the Analyst® Software 1.6.2 (AB Sciex Pvt. Ltd, USA).
Data were expressed as range and median. The Shapiro–Wilk test was performed for checking the normality. Based on normality, either Wilcoxon rank-sum test (Mann–Whitney U test) or unpaired t-test was used to compare the PHT concentration in all three matrices. P ≤ 0.05 was considered statistical significant. The R software (version 3.4.4) was used for statistical computation. The additional packages i.e., ggplot2 and ggsignif  were also used.
In both control and aSAH groups, the median PHT concentration in all three matrices was calculated for comparison. In addition, the brain and CSF PHT concentrations were computed as a fraction of plasma PHT concentration using the following formula. Hypothetically, 1 ml of plasma was equated to that of 1 μg of brain tissue for calculation.
Brain PHT concentration as fraction of plasma = (PHT concentration in brain tissue/PHT concentration in plasma) * 100
CSF PHT concentration as fraction of plasma = (PHT concentration in CSF/PHT concentration in plasma) * 100
This is a pilot study comparing the alteration in PHT permeability across BBB in aSAH grade 3 or 4 (with apparent BBB disruption) versus the control (tumor) with no radiological evidence of BBB disruption. There were 10 subjects enrolled in each group (control and aSAH). As the PHT concentration in brain tissue in one of the subjects in aSAH group was found to be an outlier (29.94 μg/g) as assessed by boxplot, the subject value was excluded for result analysis from all matrices. The final analysis was done between control (n = 10) and aSAH group (n = 9). For control group, the median concentration of PHT was found to be as follows:
For aSAH group, the median concentration of PHT was found to be as follows
The distribution of data for some matrices was found to be nonparametric (as assessed by Shapiro–Wilk test; P < 0.05). The Mann–Whitney U test was used for the comparison of results between control and aSAH group. Median PHT concentration in plasma for control (11.59μg/ml) and aSAH (10.82μg/ml) was not significantly different, P = 0.661 [Figure 1]. Median PHT concentration in CSF for control (1.07μg/ml) and aSAH (0.45μg/ml) was not significantly different, P = 0.4377 [Figure 1]. Median PHT concentration in brain tissue was significantly higher in the control (10.73 μg/g) than in the aSAH group (3.78 μg/g), P = 0.010 [Figure 1]. Median PHT concentration as assessed by the CSF concentration as fraction of plasma for control (7.37 μg/g) and aSAH (4.34 μg/g) was not significantly different, P = 0.4967 [Figure 2]. Median PHT concentration as assessed by the brain concentration as fraction of plasma was significantly higher in control (89.55 %) as compared to aSAH group (36.72 %), P = 0.003 [Figure 2].
The demographic parameters were similar for the aSAH and the control group. Subjects having aneurysmal subarachnoid bleed of grade 3 and 4 (different sites) were enrolled in aSAH group. To evaluate the alteration in BBB permeability of PHT in the aSAH group, we compared them with the control group consisting of subjects with astrocytoma, having no obvious disruption of BBB  evidenced by noncontrast-enhancing intracerebral mass lesions. The results were computed as median PHT concentration in all three matrices (plasma, CSF, and brain tissue) in both groups [Figure 1]. In addition, the PHT concentration in brain and CSF was computed as a fraction of plasma to standardize PHT concentration in these matrices according to the variation observed in the plasma [Figure 2].
No significant difference was found in plasma median PHT concentration in the aSAH group as compared to control group. The estimated median PHT concentration in plasma of control and aSAH were found to be 11.59 μg/ml and 10.82 μg/g, respectively, and the values were in accordance with available literature., Lower PHT concentrations were observed in CSF as compared to plasma and brain tissue [Figure 1]; this is well known and is due to the lower content of protein in the CSF. On comparing median PHT concentration in brain of aSAH with that of controls, PHT was found to be significantly decreased in aSAH group as compared to the control group (64.8%, P = 0.010) [Figure 1]. Similarly, when PHT concentration in brain tissue (calculated as a fraction of plasma) was compared between both control and aSAH groups, there was a significant decrease in PHT concentration in aSAH group (59%, P = 0.003) [Figure 2]. This clearly demonstrates that the concentration of PHT penetrating the brain following aSAH is hampered and there is a significant decrease in its permeability across BBB after aSAH in grade 3 and 4. Hence, the utility of PHT administration as a prophylactic antiepileptic in cases of aSAH seems questionable. Further, the electrolyte abnormalities in aSAH could contribute to the seizurogenic activity rather than cortical damage and hence the correction of imbalances should gain priority in clinical management.
This is the first study that demonstrates the proof with laboratory evidence of decreased penetration of PHT across BBB in aSAH of grade 3 and 4 patients. The results are in agreement with previous study conducted in rodents, where similar findings were obtained in brains after experimental BBB disruption. The previous literature revealed that damaged BBB affects the distribution of lipophilic drugs like that of PHT where the concentration of drugs reaching into the brain is decreased. In our study, we also found that a definite reduction in PHT concentration reaching the brain parenchyma in subjects with aSAH compared to those with cerebral tumors, where there is no obvious BBB disruption.
Cerebral vasospasm is one of the important events following aSAH that accounts for poor prognosis in these patients. In the current study, the brain tissue obtained was peripathological in both tumors (controls) and aSAH (cases); thus, the effect of edema on the BBB permeability and its effects on drug penetration was not evaluated in this study and remains a question for further research. Moreover, the reliability of the results can be tested in prospective studies correlating the radiological parameters and differential distribution of the drug in edematous and nonedematous areas of the brain.
Limitations of the study
We conclude that there is a definite decrease in PHT permeability across BBB following aSAH grade 3 and 4. This decreased penetration makes us wary of continued use of prophylactic PHT in in aSAH management, especially when the said drug has been notorious for long-term morbidity, including chest infection and cerebral infarcts. Further studies incorporating a larger sample size are warranted to support or refute the role of altered BBB permeability of PHT in aSAH.
Ethical conduct of research
The approval of the Institute Ethics Committee (NK/1791/Study/2173) was obtained before conducting the study.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
[Figure 1], [Figure 2]
[Table 1], [Table 2]