Animals
All studies were performed according to protocols approved by the Institutional Animal Ethical Committee (Approval no. 107/99/CPCSEA -2016-08), Punjabi University, India. Adult female Swiss albino mice (19-23g, 10-12 weeks old) were used for these studies, four to five mice were kept in a cage, mice had ad libitium access to food and water, and they were maintained on a 12h light/dark cycle.
Drugs and chemicals
Pregnant mare’s serum gonadotropin (PMSG), human chorionic gonadotropin (HCG), finasteride, β-Cyclodextrin, estradiol, progesterone, pentylenetetrazole, ferulic acid, dopamine, serotonin and methanol (HPLC grade) were obtained from Sigma (USA), Heptane sulfonic acid (Merck, India), perchloric acid and tartaric acid (Spectrochem, Mumbai), norepinephrine (Troikaa Pharmaceuticals, India). Sodium-L-glutamate (S D Fine-Chem limited, India), trichloroacetic acid and pyridoxal-5-phosphate (Sigma Chemical Co., St. Louis MO, USA), ninhydrin and gamma-aminobutyric acid (GABA) (Sigma Chemical Co., St. Louis MO, USA), triton X-100 (scintillation grade) (Loba Chemie) were also used in the study. All other chemical reagents were of analytical grade.
Neurosteroid withdrawal model of catamenial epilepsy and seizure susceptibility
A state of prolonged high serum progesterone level (pseudopregnancy) was induced in mice by sequential injection of pregnant mares' serum gonadotropin PMSG (5 IU s.c.) followed 46 h later by human chorionic gonadotropin HCG (5 IU s.c.) (Reddy et al. 2001). The day of the second gonadotropin injection was considered day 0. Neurosteroid withdrawal was induced by treatment with finasteride (50 mg/kg, i.p.), a 5α-reductase inhibitor on day 9 that blocks the conversion of progesterone to allopregnanolone, decreased neurosteroid levels, mimicking perimenstrual changes in women.
Animals were randomly divided into six cohorts (n=6 each). Cohort I (Naïve group), cohort II to VI were administered PMSG followed by HCG 46 h after, followed by finasteride (50 mg/kg, i.p.) on day 9. Cohort II and III were labelled as pseudo pregnant (positive control) and finasteride (negative control) groups, respectively. Following PMSG and HCG, cohort IV to VI also received ferulic acid (25, 50 and 100 mg/kg i.p.) for 10 days. The cohort II to VI received finasteride (40 mg/kg, i.p.) on day 9 of treatment and to assess seizure severity subconvulsant PTZ dose (40 mg/kg, i.p.) was given on day 10 (24h after finasteride injection) (Figure 1) (12). Latency to clonic seizures, latency to generalized tonic-clonic seizures (GTCS) as well as seizure severity was observed according to modified Racine’s scale, Stage 0: no response, Stage 1: hyperactivity, restlessness and vibrissae twitching, Stage 2: head nodding, head clonus and myoclonic jerks, stage 3: unilateral or bilateral limb clonus, Stage 4: forelimb clonic seizures, Stage 5: generalized tonic–clonic seizures with falling, Stage 6: hind limb extensor and death was considered as Stage 7. Animals failing to show clonic spasms which are characterized by the rapid involuntary rhythmic contraction and relaxation of limbs lasting longer than 5 s were scored as protected. Animals failing to show clonic spasms (rapid involuntary rhythmic contraction and relaxation of limbs) lasting longer than 5 sec were considered protected and scored 0. Seizure scoring was performed live by one of the authors (TS) in a blinded manner. Four to six hours after PTZ injection (animals restore normal behavior two hours following PTZ injection), animals were evaluated for depression-like phenotypes using a tail suspension test (only once on treatment day 10). Four hours following TST, animals were anesthetized, chest cavity was cut opened to collect blood directly from heart and brain sub regions (cortex and hippocampus) were harvested for estimation of monoamines. We have been using PTZ model (acute as well as chronic) in our lab for over a decade to study various phenomenon related to epileptogenesis, and we are pioneers in using post kindled animals for depression and cognitive comorbidities of epilepsy. Our pilot studies have shown that 4h after tail suspension test, circulating hormones and cerebral monoamine levels return to baseline, which explained animal euthanasia on these time intervals.
Tail suspension test
The tail suspension test was conducted as previously described (Singh et al. 2017), with some modifications on day 10. Briefly, mice were individually suspended by tail with a clamp (1 cm from the tip of the end). A mouse was suspended for a total of 6 min, and the duration of immobility was recorded during the final 4 min interval of the test. Mice were considered immobile only when they hung passively and completely motionless.
Determination of serum neurosteroids levels
Neurosteroids were estimated in serum samples using previously reported HLPC-UV method (Wei et al. 1990) with slight modifications. To prepare sample, serum (10 µl) was digested with ethyl ether (1 ml), vortexed for 3 min and then centrifuged at 3500 rpm for 5 min. The organic layer was transferred to a test tube and evaporated to dryness at 50 ⁰C. Before injection, 50 µl mixture of methanol and water (60: 40 v/v) was added, vortexed and vibrated ultrasonically for 60 s. After centrifugation for 2 min at 4000 rpm, 20 µl of supernatant was injected into the system.
Waters HPLC system (Milford, USA) consisted of 515 binary pumps (Waters, USA), 2489 ultraviolet detector (Waters, USA) and rheodyne manual injector (20μl) was used. The chromatographic separation performed at room temperature was achieved using Zorbex SB-C18, reversed phase column (4.6 mm x 150 mm x 5 µm) (Agilent, USA) at 254 nm. The mobile phase consisted of a mixture of methanol: tetrahydrofuran: water (26:18:56 v/v/v) with a flow rate of 1 ml/min at room temperature, filtered using 0.45 µm membrane (Millipore, USA) and degassed using Transonic T 570/H, Elma, Germany. The data was acquired and processed in Empower Pro® Operating System (Waters®, Milford, USA). A stock solution of corticosterone, estradiol and progesterone were prepared in 1 mg/mL methanol and standard curve was plotted. Corticosterone, y = 44261x + 13104, R2 = 0.996, estradiol, y = 0.735 x + 0.905, R² = 0.987, and progesterone, y = 63171x - 38604, R² = 0.999). The data were acquired and processed in Empower Pro Operating System (Waters, Milford, USA).
Neurochemical estimations
Monoamines (norepinephrine, dopamine, and serotonin) were estimated using HPLC-ECD as reported previously by our lab (Singh et al. 2016) and another half was used for estimation of total nitrite levels using microplate reader, also reported previously (Singh et al. 2015).
Glutamic acid decarboxylase (GAD) Activity
GAD activity was assayed according to the method reported (Wolf and Klemisch 1991). Enzyme activity was expressed as micro-gram GABA/ mg protein of wet tissue.
Statistical Analysis
The statistical analysis was performed using Graphpad prism® version 8 (Graph-Pad Software Inc., San Diego, CA, USA). Statistical significance was calculated using one-way ANOVA followed by Student-Newman-Keuls test. Each value was expressed as mean ± SEM, and statistical significance was considered at p < 0.05.