Intervention

Hundred patients were recruited for the study. Five groups of patients were compared. Informed written consent was taken. Patients in each group received 150 mg aspirin. The control group (N) received supportive care. Each group had 20 patients of either gender. The drug citicoline is an exogenous form of cytidine-5‘-diphosphocholine needed for membrane biosynthesis. It acts as a membrane stabilizer and reduces free radical formation thus reducing ischemic injury.5 The citicoline dose used was 500 mg twice daily for 6 weeks either as parenteral or oral formulation. Edaravone is also free radical scavenger that delays neuronal death, prevents vascular endothelial injury, inhibits in vitro lipoxygenase pathway activation in the arachidonic acid cascade and phosphatidylcholine liposomal membrane peroxidation, thereby reducing ischemic brain edema.6 It was administered as intravenous infusion at a dosage of 30 mg twice daily over 60 minutes for 14 days. Minocycline is lipophilic drug that crosses the blood–brain barrier (BBB), inhibits microglial activation, reduce T cell migration and reduces neuronal apoptosis and generation of free radicals and chemokines and their receptors expression in the central nervous system.7 Minocycline was administered as oral formulation in a dose of 200 mg/day for 10 days. The drug cerebrolysin is a porcine brain-derived free amino acids and low-molecular-weight neuropeptides (10 kDa).8 Cerebrolysin was administered as intravenous infusion in a dose of 30 mL diluted in 100 mL saline over 60 minutes for 10 days.

Clinical Characteristics

The demographic details, clinical symptomatology, presence of diabetes mellitus, hypertension, ischemic heart disease, dyslipidemia, atrial fibrillation, rheumatic heart disease, alcohol, and nicotine abuse, time of onset of symptoms to NPA administration, if thrombolytics was used and NIHSS at admission, at day 11 and after 90 days, were recorded. The location of infarct (right or left) and Alberta Stroke Program Early CT score (ASPECTS) score was calculated in each patient.

Investigations

The following blood investigations were performed in all patients that include complete blood counts, prothrombin time and international normalized ratio, activated plasma thromboplastin time, renal function test, serum electrolytes, random blood sugar, glycated hemoglobin, fasting lipid profile, serum homocysteine, human immunodeficiency virus, hepatitis B virus, and syphilis serology. MRI brain including diffusion-weighted imaging in 1.5-Tesla and CT brain were done in every patient.

Outcome

The clinical assessment to record the functional outcome was done at discharge and 90 days. Barthel index (BI) and mRS were employed to assess the functional outcome. The mRS score and BI were recorded at admission, day 11 and after 90 days. The hospital mortality and its causes were also included.

Statistical Analysis

Student’s unpaired t-test was used to compare continuous variables expressed as mean ± standard deviation. Chisquared test was used to compare categorical variables expressed as frequency with percentage. Analysis of variance tests and multiple comparison tests were used for comparison of NIHSS, mRS, and BI scores among individual NPA and placebo group. Mann–Whitney U tests were used to determine a significant change in mRS score at day 11 and after 90 days. Kruskal–Wallis tests were used to determine the existence of statistically significant differences among different groups with change in mRS score at day 11 and after 90 days. p-Value ≤ 0.05 was considered as statistically significant. The statistical analyses were conducted using SPSS 17 software (IBM. Inc., Texas, United States).

Results

One hundred patients were included in the study with 59 males and 41 females. The mean age of the patients was 58.4 ± 7.2 (range: 50–67) years. They were 20 patients each in citicoline (C), edaravone (E), minocycline (M), cerebrolysin (Cs), and fifth control group (N). There were 12 males and 8 females in citicoline group; 11 males and 9 females in edaravone group, 13 males and 7 females in minocycline group, 12 males and 8 females in cerebrolysin group, and 11 males and 9 females in control group. Each group was matched for age, gender, time of initiation of treatment, preexisting medical conditions, mRS/NIHSS, and ASPECTS score at the time of admission. The mean time until admission after onset of stroke symptoms and meantime until treatment with NPA administration was similar among all the groups with no statistical significance. Nineteen patients received thrombolysis treatment with no statistically significant difference among the groups. The demographic details and clinical profile of the three groups are shown in Table 1.

There was reduction in mean NIHSS score at day 11 and after 90 days in citicoline, edaravone, and cerebrolysin group in comparison with placebo assuming statistical significance (Table 2). However, in minocycline group, the NIHSS score did not reduce at day 11 and after 90 days. Similarly, the mean BI score was significantly higher at day 11 and after 90 days in citicoline, edaravone, and cerebrolysin group in comparison with placebo (Table 3).

Safety Analysis

The adverse effects of neuroprotective agents were recorded every day during the stay in hospital till day 11. No adverse effects were noted in any of the group except two patients in cerebrolysin group who had a fever that resolved with antipyretics.

Analyses of Mortality

There were five deaths with one death in each group. The cause of death in edaravone and minocycline group was due to malignant MCA territory infarct with herniation (despite decompressive craniectomy). Other three deaths occurred following discharge within after 90 days (cause not known).

Discussion

Agents that are easy to administer, ability to reduce tissue damage during AIS, will help in improving functional outcomes and quality of life of patients. One of those agents is the neuroprotective agents. These agents protect the brain from ischemia in AIS.9 Antithrombotics, antiplatelets, and thrombolytics also produce neuroprotection by targeting the cerebral vessels and are considered as extrinsic or indirect neuroprotectants. Direct neuroprotectants act directly on the neuron. Brain ischemia causes disruption of the BBB and loss of microvascular integrity as it triggers extracellular and intracellular proteolytic cascades.10 During reperfusion, there is reactive oxygen species (ROS) production that causes ischemic cells to secrete inflammatory cytokines and chemokines that increase adhesion molecules and cause recruitment of peripheral leukocytes. The recruited inflammatory cells release more cytokines, matrix metalloproteinases (MMPs), nitric oxide, and more ROS. MMPs cause BBB disruption, activation of microglia, and recruitment of peripheral inflammatory cells.11 The pharmacological modulation of molecular targets in the ischemic cascade can produce neuroprotection.9 These targets include glutamate release and glutamate receptor activation, excitotoxicity, calcium influx into cells, mitochondrial dysfunction, and activation of intracellular enzymes, free radical production, nitric oxide production, apoptosis, and inflammation. We used four neuroprotective agents in our study. The objective of our study was to determine the effectiveness of four neuroprotective agents (citicoline, edaravone, minocycline, and cerebrolysin) in changing the functional outcome at 90 days of patients with AIS involving MCA territory. All the patients in our study had moderate-to-severe stroke with mean NIHSS score of 14.

Minocycline is an antibiotic belonging to the tetracycline family and has anti-inflammatory and neuroprotective action. It is a ROS scavenger protecting brain tissue against oxidative stress, which inhibits microglial activation and caspase-1, caspase-3, cyclo-oxygenase-2, inducible nitric oxide synthase, p38 mitogen-activated protein kinase, and MMP-9.12 A study by Lampl et al, on minocycline usage in AIS, showed beneficial effect in minocycline-treated group with significant improvement in NIHSS score.13 A study by Fagan et al showed that with intravenous minocycline (no controls), 50% (60 patients) of patients had mRS of 0 or 1 at 90 days.14 A study by Padma Srivastava et al also showed significant improvement in NIHSS score, BI score, and mRS score with minocycline.15 A study by Amiri-Nikpour et al showed a similar decrease in NIHSS in patients receiving minocycline at 90 days.16 However, study by Kohler et al did not find any benefit with minocycline.17 The dose used was 100 mg/ day, half of what the other studies had used, and was continued only for 2.5 days. In our study, 20 patients received oral minocycline at a dosage of 200 mg/day for 5 days similar to the above studies. The mean time between stroke onset and minocycline administration was 12.4 hours. In the other studies, it was from 5 to 24 hours. There was decrease in NIHSS score and improvement in BI score in minocycline–treated group and control group but was not statistically significant. Minocycline was not effective as compared with control in improving the functional outcome.

Cerebrolysin is a neuropeptide and has neuroprotective properties. It reduces excitotoxicity and inhibits free radical generation, microglial activation/neuroinflammation, and calpain activation/apoptosis. It has neurotrophic activity as it promotes neuronal sprouting, improves cellular survival, and stimulates neurogenesis. Cerebrolysin acute stroke treatment in Asia trial did not show a significant difference in the functional outcome between the cerebrolysin and placebo groups, but there was a beneficial trend in favor of cerebrolysin on post-hoc analysis.18 Cerebrolysin and recovery after stroke trial showed that patients treated with cerebrolysin had an improvement in the NIHSS on day 90 compared with placebo.19 Study by Xue et al on cerebrolysin in AIS showed significant improvement of 21-day NIHSS scores with cerebrolysin.20 In our study, 20 patients received intravenous cerebrolysin with mean baseline NIHSS score of 14.15 ± 5.3, and there was a significant improvement in the NIHSS and BI score at day 11 and after 90 days.

Edaravone is an antioxidant and is low molecular weight lipid-soluble free radical scavenger that readily crosses the BBB. Edaravone AIS Group (Otomo clinical trial report) showed that edaravone administration resulted in significant clinical improvement.21 A study by Sinha et al showed that patients in edaravone-treated group had significant reduction in NIHSS and improvement in BI at 90 days.22 Edaravone—Citicoline Comparative Study in AIS by Mitta et al—showed that edaravone usage resulted in a better outcome at 90 days in patients with AIS.23 In our study, 20 patients received intravenous edaravone with mean baseline NIHSS score of 13.2 ± 5.3 with significant improvement in the NIHSS and BI score at day 11 and after 90 days.

Citicoline helps in restoring the activity of mitochondrial ATPase and membrane Na⁺/K+ ATPase, inhibits the activation of phospholipase A2, and accelerates the reabsorption of cerebral edema.24 The International Citicoline Trial on Acute Stroke study showed similar global recovery at 90 days in citicoline and placebo group. The study did not provide evidence that citicoline is efficacious in moderate-to-severe AIS.25 A meta-analysis by Saver (10 controlled clinical trials that used citicoline) showed that citicoline caused a significant decrease in the frequency of death or disability at follow-up.26 Study by Ghosh et al reported favorable outcome and an increased probability of complete recovery following citicoline treatment in all types of strokes.27 In our study, 20 patients received intravenous/oral citicoline with mean baseline NIHSS score of 14.0 ± 4.3 with significant improvement in the NIHSS and BI score at day 11 and after 90 days.

The small sample size in each group was the limitation of our study. It was a single-blinded study wherein the investigators were aware about the neuroprotective agents received by the patients and the outcome was assessed by the investigators themselves with a possibility of research bias. A randomized control trial with a bigger sample size is required to strengthen the results of this study.