Estradiol does not affect spasms in the betamethasone-NMDA rat model of infantile spasms

Abstract

Objective: This study attempted to validate the effects of neonatal estradiol in ameliorating the spasms in the prenatally betamethasone-primed N-methyl-d-aspartate (NMDA) model of infantile spasms in rats as shown previously in a mouse Arx gene knock-in expansion model of infantile spasms.

Methods: Neonatal rats prenatally exposed to betamethasone (on day 15 of pregnancy) were treated with subcutaneous 40 ng/g estradiol benzoate (EB) between postnatal days (P)3-P10 or P0-P5. A synthetic estrogen analogue, diethylstilbestrol, was used between P0 and P5 (2 μg per rat, s.c.). On P12, P13, and P15, the rats were subjected to NMDA-triggered spasms, and latency to onset and number of spasms were evaluated. Rats with EB on P3-P10 were tested after spasms in the open field, novel object recognition, and elevated plus maze to determine effects of treatment on behavior. Additional rats with P3-P10 or P0-P5 EB were investigated for γ-aminobutyric acid (GABA)ergic neurons (glutamate decarboxylase [GAD]67 expression) in the neocortex. As a positive control, a group of rats received either subcutaneous adrenocorticotropic hormone (ACTH) (2 × 0.3 mg/kg on P12 and 3 × 0.3 mg/kg on P13 and P14) or vehicle after the first episode of spasms on P12.

Results: Neither EB treatment nor diethylstilbestrol consistently affected expression of spasms in this model, although we found a significant increase in GAD67-immunopositive cells in the neocortex after P3-P10 and P0-P5 EB treatment, consistent with a study in mice. Behavioral tests showed increase in lateralization in male rats treated with P3-P10 EB, a behavioral trait usually associated with female sex. Diethylstilbestrol treatment in male rats resulted in arrested pubertal descent of testes. ACTH had robust effects in suppressing spasms.

Significance: Treatment of infantile spasms (IS) using neonatal EB may be justified in those cases of IS that present with detectable deficits in GABAergic neurons. In other types of IS, the efficacy of neonatal EB and its analogues is not supported.

Keywords: ACTH; Behavior; Diethylstilbestrol; Epileptic spasms; Validation.

Figure 1.

Effects of neonatal (P3-P10) treatment with estradiol benzoate (EB) on the expression of spasms, number of GAD67 immunopositive cells in the neocortex, and body weight gain Controls were injected with oil between P3-P10, EB rats received 40 ng/g body weight of EB daily on P3-P10. There was never main effect of sex and no interaction of sex with treatment; therefore the factor of sex was removed from all analyses. All values refer to mean±SEM. (A) Latency to onset of the first occurrence of spasm on P12, P13, and P15 day of spasms. No significant effect was found. (B) Latency to onset of the fully developed spasm on P12, P13, and P15. No significant effect was found. (C) Number of spasms per observation period on P12, P13, and P15. No significant effect was found. (D) Scheme for counting neurons in the neocortex. Three brain sections at matching positions were selected from each animal (80 μm apart; not to scale). Three rectangles (150 μm wide) were placed on the neocortex in each section spanning its entire depth. Images were calibrated for distance so the height of each column was measured. All neurons within columns were counted and their density averaged per animal. The average entered statistical evaluation (each animal was a unit). Shaded parts of rectangles represent counting layers V-VI. (E) Example of the neocortical cross-section with GAD67 immunopositive cells in the rat treated with oil between P3-P10. Note few immunopositive cells in the section. Lens magnification 4x. Scale bar is 200 μm. (F) Example of the neocortical cross-section with GAD67 immunopositive cells in the rat treated with EB between P3-P10. Note numerous immunopositive cells in the section. Lens magnification 4x. Scale bar is 200 μm. (G) Number of GAD67 immunopositive cells in the sampled area of sensorimotor cortex. N of subjects in the EB group = 6; n of subjects in the oil group = 6. Left, counting neurons throughout the cortex, EB induced significant increase compared to oil, *p=0.01. Right, counts within layers V-VI, again there was a significant increase after EB compared to oil, *p=0.01. None of these animals were subjected to spasms. (H) Body weight gain between P1 and P15. EB-treated rats gained significantly less weight compared to oil-injected, *p=0.005.

Figure 2.

Performance of the P22-P24 rats in the Novel Object Recognition test after neonatal (P3-P10) treatment with EB and three bouts of spasms on P12, P13, and P15. All values refer to mean±SEM. Group sizes are oil-male n=5; oil-female n=6; EB male n=5 EB female n=6. (A) Scheme of the experiment (left-timing; right-arena). For each animal, there were 5 test sessions separated with 1 hour. First three sessions used objects A1 on the left (L) and A2 on the right (R, these were always the same bowling pins). Data from these sessions were averaged for baseline indices. Note that negative numbers in the indices indicate preference for the left side object, positive numbers for the right side objects. The fourth session used objects A1 (L) and a novel object B (R). Finally, in the fifth session, the pin was replaced for a different pin from the same set A3 (L) and another novel object C was placed on the position R. Scheme of the arena for the experiment. We used rectangular arena of size 45.5 × 45.5 cm with 30 cm high transparent walls. L and R indicate positions of the objects shown on the left. The cartoon rat represents starting position for each trial. (B) Preference index for contacts with objects (number of contacts). Baseline represents mean values from three separate trials with identical objects A1 and A2. In the graph “1st novel object” depicts the preference for the novel object (B) always placed in the right position, while object A1 was kept on the left. The graph “2nd novel object” shows the preference for another novel object (C, right) in comparison to the object A3 (left), which is similar to A1/A2 objects. There was an effect of treatment on baseline performance; *p=0.02. (C) Preference index for exploration of objects (time spent exploring the objects). There was an effect of treatment in the baseline performance *p=0.002. Other details as above. (D) Curiosity index (composed of the latency to onset of exploration and duration of exploration of the particular object). There was a trend in the effect of treatment during baseline testing #p=0.08. Other details as above.

Figure 3.

Performance of the rats in the Elevated Plus-Maze test (EPM; P31-P32) and in the Open Field test (OF; P21–22) after neonatal (P3-P10) treatment with EB and three bouts of spasms on P12, P13, and P15. Since sex as a factor had no significant effect on performance and there was no interaction with treatment, males and females were pooled. All values refer to mean±SEM. (A) Elevated Plus Maze. Left side – duration of exploration of the open arm, central area and the closed arm. Right side – ratio between time spent in the distal half of the open arm over time spent in the distal half of the closed arm. No effect of treatment was found. (B) Central zone of the open field (OF CENTRAL): From left – total time spent in the central zone out of 300 s test time, time spent ambulating, time spent with stereotypies, time spent in resting (all in seconds). Right – total distance traveled in the central zone (in cm). No effect of treatment was found. (C) Peripheral zone of the open field (OF PERIPHERAL): From left, total time spent in the zone is omitted, otherwise details as above. (D) Left, arrangement of the zones in the open field with the representation of the central area (20.75% out of the total area) and time spent in the central area as a proportion of total test time (4.2%; i.e., five times less than the area proportion out of total open field is). Right, time spent in the central zone as a proportion of total test time in the open field broken down by treatment and sex.

Figure 4.

Effects of early neonatal (P0-P5) treatment with EB or diethylstilbestrol (DES) We never detected main effect of sex or interaction of sex with the factor of treatment. Therefore, male and female data were pooled. Number of subjects in each subgroup was between 7 and 25. All values refer to mean±SEM. (A) Latency to onset of the first occurrence of spasms on P12, P13, and P15 test days. DES delayed onset of the first spasm during P12 testing; *p=0.04. (B) Latency to onset of the fully developed spasms on P12, P13, and P15. EB delayed latency to onset of fully developed spasms on P15; *p=0.03. (C) Number of spasms per observation period on P12, P13, and P15. There was a trend to decreased number of spasms on P12 after DES treatment; *p=0.05 (Kruskal-Wallis test corrected for ties). (D) Relative numbers of GAD67-immunopositive cells in the neocortex. Early neonatal (P0-P5) EB increased number of GAD67 cells by 21.0% compared to oil throughout neocortex (*p=0.002). Counts only in layers V and VI revealed smaller, yet significant (*p=0.02) increase by 17.3% compared to oil. (E) Body weight gain between P1 and P15. Overall significance; ANOVA p<0.0011. Effect of EB treatment vs. oil; *p=0.04. Effect of DES treatment vs. oil; *p<0.001. (F) P31 male (time of puberty onset) neonatally injected with DES. Note that the testes still remain in the abdominal cavity (no descent). The scrotum is not formed (arrow). (G) P31 male neonatally injected with oil. Descended testes in the scrotum are clearly visible (arrow) compared to (C).

Figure 5.

Effects of ACTH in the randomized prospective trial on the expression of spasms First bout of spasms was triggered on P12 and afterwards the rats were randomized to the treatment (ACTH) or vehicle groups (efficacy of randomization is shown in the P12 column in A, B, C; blue arrow marks randomization). Afterwards they received ACTH 0.3 mg/kg per dose s.c. twice on P12 and three times on P13, and P14. Spasms were triggered on P13 and again on P15 (12–14 hours after the last ACTH dose). There were consistent effects of ACTH on the expression of spasms despite smaller n of subjects (5–10 in each subgroup). (A) Latency to onset of the first occurrence of spasms on P12, P13, and P15 test days. There was an effect of ACTH in delaying onset of the first spasm during P15 testing; *p=0.02. (B) Latency to onset of the fully developed spasms on P12, P13, and P15. We found an effect of ACTH again in delaying latency to onset of fully developed spasms on P15; *p=0.01. (C) Number of spasms per observation period on P12, P13, and P15. There was a significant decrease in number of spasms on P15 after ACTH treatment; *p=0.02. (D) Body weight gain between P1 and P15. There was no effect of ACTH treatment. (E) Body weight gain during the ACTH treatment period P12-P15. There was a trend to decreased body weight gain after the ACTH treatment; #p=0.08.