Targeting CRHR1 Signaling in Experimental Infantile Epileptic Spasms Syndrome: Evidence for Route-Dependent Efficacy methods
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2. Materials and Methods
All animal experiments were approved by the IACUC of the New York Medical College and complied with the NIH Guide for the Care and Use of Laboratory Animals, 8th edition. Timed pregnant Sprague-Dawley rats were purchased from Taconic Biosciences (Germantown, NY, USA). and delivered to the institutional Animal Facility on day 8 of pregnancy (G8). They were housed individually on a regular 12 h light/12 h dark cycle (lights on at 07:00 h) with free access to rat chow and tap water. After one week of acclimation, on G15, pregnant females were injected with betamethasone (betamethasone phosphate, MilliporeSigma, St. Louis, MO, USA, in normal saline, 2 × 0.4 mg/kg intraperitoneally = i.p.) at 08:00 and 18:00 h [ ].
2.3. Surgeries
On P13, rat pups (both males and females) were anesthetized by isoflurane inhalation (induction: 5% in oxygen in the induction chamber; maintenance: 3% in oxygen through the stereotaxic instrument mask) and fixed to the stereotaxic instrument (David Kopf Instruments, Tujunga, CA, USA). The skin from the skull was removed, and the fascia was cleaned. Surface bleeding was stopped with 3% hydrogen peroxide. A position for the microinfusion guide cannula (Plastics One, now Protech International, Inc., Boerne, TX, USA) was set according to the anteroposterior and lateral coordinates (, with bregma set to zero). A small hole was drilled into the bone, and the guide cannula was lowered to the set depth. The guide cannula coordinates were set 1 mm shallower than the target structure coordinates, since the microinfusion cannula extended 1 mm beyond the guide cannula. A single guide cannula wa...
2.4. Intracranial Drug Microinfusions
On P15, the animals were gently immobilized in hand, and the microinfusion cannula was carefully inserted through the implanted guide cannula to the structure of interest (lateral ventricle, third ventricle, or arcuate nucleus). Solutions were microinfused using a 5 µL Hamilton microsyringe (Reno, NV, USA) attached to a Teflon tube. Drug solutions at varying concentrations ( ) were prepared in a fixed total volume of 0.5 µL and were slowly microinfused over 1 min; vehicle controls received 0.5 µL of vehicle over 1 min. After microinfusing, the cannula stayed in place for an additional 1 min before being retracted to prevent backflow of the solution [ ]. Both CP376395 and SN003 microinfusions were completed 1 h prior to NMDA induction of spasms.
2.5. Systemic Drug Administration
On P15, both drugs were injected i.p. 1 h prior to the NMDA induction of spasms. CP376395 was diluted in normal saline and injected at 3 mg/kg, whereas SN003 was first diluted in 100% ethanol, then diluted with distilled water to a 1% ethanol solution and injected at 0.7 mg/kg. Controls always received equivalent volumes of respective vehicles. Drug doses were derived from published doses used for in vivo experiments in adult rats, namely, systemic CP376395 of 5.0-10.0 mg/kg [ ] and systemic SN003 of 0.5-1.0 mg/kg [ ].
2.6. NMDA Trigger of Spasms
NMDA was administered i.p. on P15 at a dose of 15 mg/kg dissolved in normal saline. Immediately after the i.p. injection, the animals were placed in separate cages on a heating pad and observed for symptomatology of NMDA syndrome. Besides marking latency to onset of tail twisting (snake-like tail movements starting at the tip), arching, and flexion spasms (emprosthotonus, hyperflexion position on the side lasting < 10 s), we also counted the total number of spasms within the 90 min observation period. Our primary outcome was the number of spasms, with a secondary outcome of latency to onset of spasms from the time point of NMDA injection.
2.7. Quality Control, Histology, and Statistics
All experimenters were blinded to whether animals received drug versus vehicle microinfusion. Every animal group in the main experiments was combined from at least 3 litters, and no more than 2 males and 2 females from a single litter were used. The animals were assigned to the groups using stratified randomization, i.e., randomization that accounted for the litter origin and sex. We also monitored the animals' body weights as a simple measure of even group randomization, and there was no difference in the body weight distribution among the groups. After the experiments, all animals with intracranial cannulas were euthanized under ketamine/xylazine (7/70 mg/kg i.p.) anesthesia and brains were extracted. Brains were flash frozen, sectioned using a cryostat, and stained with cresyl violet (Nissl stain) for microscopic verification of the tip of the cannula placement. Only animals...
3.2. Targeted Brain Intraparenchymal Microinfusions of the CP376395
Our previous data indicated that the hypothalamic arcuate nucleus was significantly involved in the expression of spasms [ ]. CRHR1 is highly expressed in the arcuate nucleus [, ]. Hence, we were interested in whether direct microinfusions of CP376395 into the arcuate nucleus would influence spasms. Notably, 1 µM CP376395 delayed the latency to onset of spasms (Student's t -test; t = 2.365; 11df; * p = 0.0375; A) and suppressed the number of spasms (Mann-Whitney U = 7.000; tied * p = 0.0280; B). To assess site specificity, we analyzed off-target microinfusions (misses) that ended in the mamillary bodies, which have lower CRHR1 expression [ ] and are not implicated in NMDA-induced spasms [ ]. There was no effect of CP376395 microinfusions (1 µM group n = 4; 1 mM group n = 3; control group n = 3) on the latency to onset of spasms (Student's t -test t = ͨ...
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All animal experiments were approved by the IACUC of the New York Medical College and complied with the NIH Guide for the Care and Use of Laboratory Animals, 8th edition. Timed pregnant Sprague-Dawley rats were purchased from Taconic Biosciences (Germantown, NY, USA). and delivered to the institutional Animal Facility on day 8 of pregnancy (G8). They were housed individually on a regular 12 h light/12 h dark cycle (lights on at 07:00 h) with free access to rat chow and tap water. After one week of acclimation, on G15, pregnant females were injected with betamethasone (betamethasone phosphate, MilliporeSigma, St. Louis, MO, USA, in normal saline, 2 × 0.4 mg/kg intraperitoneally = i.p.) at 08:00 and 18:00 h [ ].
On P13, rat pups (both males and females) were anesthetized by isoflurane inhalation (induction: 5% in oxygen in the induction chamber; maintenance: 3% in oxygen through the stereotaxic instrument mask) and fixed to the stereotaxic instrument (David Kopf Instruments, Tujunga, CA, USA). The skin from the skull was removed, and the fascia was cleaned. Surface bleeding was stopped with 3% hydrogen peroxide. A position for the microinfusion guide cannula (Plastics One, now Protech International, Inc., Boerne, TX, USA) was set according to the anteroposterior and lateral coordinates (, with bregma set to zero). A small hole was drilled into the bone, and the guide cannula was lowered to the set depth. The guide cannula coordinates were set 1 mm shallower than the target structure coordinates, since the microinfusion cannula extended 1 mm beyond the guide cannula. A single guide cannula was implanted into either the lateral ventricle, the third ventricle, or the arcuate nucleus. There is extensive communication between the lateral ventricles through the interventricular foramina, justifying unilateral microinfusion [ ]. After insertion of the guide cannula, two anchoring jeweler scre...
On P15, the animals were gently immobilized in hand, and the microinfusion cannula was carefully inserted through the implanted guide cannula to the structure of interest (lateral ventricle, third ventricle, or arcuate nucleus). Solutions were microinfused using a 5 µL Hamilton microsyringe (Reno, NV, USA) attached to a Teflon tube. Drug solutions at varying concentrations ( ) were prepared in a fixed total volume of 0.5 µL and were slowly microinfused over 1 min; vehicle controls received 0.5 µL of vehicle over 1 min. After microinfusing, the cannula stayed in place for an additional 1 min before being retracted to prevent backflow of the solution [ ]. Both CP376395 and SN003 microinfusions were completed 1 h prior to NMDA induction of spasms.
On P15, both drugs were injected i.p. 1 h prior to the NMDA induction of spasms. CP376395 was diluted in normal saline and injected at 3 mg/kg, whereas SN003 was first diluted in 100% ethanol, then diluted with distilled water to a 1% ethanol solution and injected at 0.7 mg/kg. Controls always received equivalent volumes of respective vehicles. Drug doses were derived from published doses used for in vivo experiments in adult rats, namely, systemic CP376395 of 5.0-10.0 mg/kg [ ] and systemic SN003 of 0.5-1.0 mg/kg [ ].
NMDA was administered i.p. on P15 at a dose of 15 mg/kg dissolved in normal saline. Immediately after the i.p. injection, the animals were placed in separate cages on a heating pad and observed for symptomatology of NMDA syndrome. Besides marking latency to onset of tail twisting (snake-like tail movements starting at the tip), arching, and flexion spasms (emprosthotonus, hyperflexion position on the side lasting < 10 s), we also counted the total number of spasms within the 90 min observation period. Our primary outcome was the number of spasms, with a secondary outcome of latency to onset of spasms from the time point of NMDA injection.
All experimenters were blinded to whether animals received drug versus vehicle microinfusion. Every animal group in the main experiments was combined from at least 3 litters, and no more than 2 males and 2 females from a single litter were used. The animals were assigned to the groups using stratified randomization, i.e., randomization that accounted for the litter origin and sex. We also monitored the animals' body weights as a simple measure of even group randomization, and there was no difference in the body weight distribution among the groups. After the experiments, all animals with intracranial cannulas were euthanized under ketamine/xylazine (7/70 mg/kg i.p.) anesthesia and brains were extracted. Brains were flash frozen, sectioned using a cryostat, and stained with cresyl violet (Nissl stain) for microscopic verification of the tip of the cannula placement. Only animals with tips in the regions of interest (lateral ventricle, third ventricle, and arcuate nucleus or mammillary body) were included in the data analysis.
Our previous data indicated that the hypothalamic arcuate nucleus was significantly involved in the expression of spasms [ ]. CRHR1 is highly expressed in the arcuate nucleus [, ]. Hence, we were interested in whether direct microinfusions of CP376395 into the arcuate nucleus would influence spasms. Notably, 1 µM CP376395 delayed the latency to onset of spasms (Student's t -test; t = 2.365; 11df; * p = 0.0375; A) and suppressed the number of spasms (Mann-Whitney U = 7.000; tied * p = 0.0280; B). To assess site specificity, we analyzed off-target microinfusions (misses) that ended in the mamillary bodies, which have lower CRHR1 expression [ ] and are not implicated in NMDA-induced spasms [ ]. There was no effect of CP376395 microinfusions (1 µM group n = 4; 1 mM group n = 3; control group n = 3) on the latency to onset of spasms (Student's t -test t = -0.37; 7df; p = 0.9718; C) or number of spasms (Student's t -test t = 0.089; 7df; p = 0.9312; D). While the power of this experiment is low, a post hoc sample size analysis determined that given the current outcome, the effect size is 0.091, requiring a sample size of 386 per subgroup to achi...
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