A new experimental rodent model of cerebral palsy and foregut dysmotility

Background: Children with cerebral palsy often suffer from inability to tolerate enteral feeds. This may manifest as retching, vomiting, abdominal pain and faltering weight (previously referred to as failure to thrive). This study has three stages the aim of the first stage was to develop and establish an animal model of cerebral palsy (CP) and foregut dysmotility so as to better understand the clinical association seen in paediatric practice and to develop new therapies for this condition in the future. The second stage involved performing an experimental technique of splanchnectomy to assess whether this could be used as a surgical tool for such patients who fail to respond to conventional therapy. The third and final stage involved an examination of human biopsies from children with CP and known foregut dysmotility. Methods: Ethical approval was obtained in accordance with the Animals (Scientific Procedures) Act 1986 (PPL 60/4262). In phase one neonatal Sprague-Dawley rat pups aged postnatal days 5-6 (P5-6) underwent midline craniotomies under anaesthesia to allow either unilateral or bilateral brain injections of the neurotoxin, ibotenic acid (IBA), in the medial prefrontal (MPFC) or insular cortex (IC) using a digital stereotaxic frame. P5-6 was chosen as this has been shown to correspond to the stage of white matter vulnerability and that neurotoxin injections at this stage can mimic lesions that appear histologically similar to human peri-ventricular white matter injury as seen in cerebral palsy. A further group underwent sham injections with normal saline. All animals were monitored with a modified APGAR score post-operatively. Gastric emptying studies were then performed between 7 and 21 days postoperatively to look for any delay in gastric emptying times (GET) and animals were weighed regularly. GET was recorded by gavaging water soluble contrast and performing time lapsed x-rays. The pups were perfused on day 28 of life and their brains and foregut were examined. Phase two then involved repeating the creation of our CP model and performing unilateral splanchnectomy via a midline laparotomy to investigate whether we could improve the GET in our model. In both phase 1 and phase 2 immunohistochemistry was performed looking at several antibodies to compounds implicated in the pathogenesis of dysmotility. In phase three, a retrospective analysis of archived human biopsies from children with CP and dysmotility was performed. Main results: In Phase 1 forty-five pups were injected in total (28 male and 17 female). The median weight at time of operation was 13.5 grams (range 7.5-21 grams). Four pups died at the start of the study two intra-operatively from haemorrhage and two in the immediate post-operative period presumably from anaesthetic complications. GET was significantly prolonged in each IBA group compared to shams, and interestingly 5 out of 6 of those with bilateral MPFC lesions demonstrated reflux during their contrast studies. In Phase 2, twenty-six rat pups were injected (14 female and 12 male). 13 pups underwent IBA injection and 13 sham injections. One pup died post brain injection from postoperative apnoea, the remainder progressed to splanchnectomy. Two pups, one from each group, died post laparotomy from intraoperative bleeding. The median GET was significantly longer in the CP model 58 minutes compared to 45 minutes (p=0.0024). Splanchnectomy reduced the GET in the CP group to 40.5 minutes (p<0.0001) Our results from phase 3 revealed that we were unable to compare our animal specimens to our cohort of archived human biopsies. The reasons for this are discussed in detail. Conclusion: In conclusion, we have developed a new and reproducible animal model of cerebral palsy and foregut dysmotility. We have shown that lesions in the brain cortex can lead to structural changes in villous and crypt architecture in the gut and that splanchnectomy improves gastric emptying in our model. Further work needed is needed to explore the mechanistic pathways and provide insight into the pathogenesis of impaired gastrointestinal motility in cerebral palsy and may lead to much needed development of new treatments.