Generation of a novel decay accelerating factor (DAF) knock-out rat model using clustered regularly-interspaced short palindromic repeats, (CRISPR)/associated protein 9 (Cas9), genome editing.

Decay accelerating factor (DAF), a key complement activation control protein, is a 70 kDa membrane bound glycoprotein which controls extent of formation of the C3 and C5 convertases by accelerating their decay. Using clustered regularly-interspaced short palindromic repeats, (CRISPR)/associated protein 9 (Cas9) genome editing we generated a novel DAF deficient (Daf-/-) rat model. The present study describes the renal and extrarenal phenotype of this model and assesses renal response to complement-dependent injury induced by administration of a complement-fixing antibody (anti-Fx1A) against the glomerular epithelial cell (podocyte). Rats generated were healthy, viable and able to reproduce normally. Complete absence of DAF was documented in renal as well as extra-renal tissues at both protein and mRNA level compared to Daf+/+ rats. Renal histology in Daf-/- rats showed no differences regarding glomerular or tubulointerstitial pathology compared to Daf+/+ rats. Moreover, there was no difference in urine protein excretion (ratio of urine albumin to creatinine) or in serum creatinine and urea levels. In Daf-/- rats, proteinuria was significantly increased following binding of anti-Fx1A antibody to podocytes while increased C3b deposition was observed. The DAF knock-out rat model developed validates the role of this complement cascade regulator in immune-mediated podocyte injury. Given the increasing role of dysregulated complement activation in various forms of kidney disease and the fact that the rat is the preferred animal for renal pathophysiology studies, the rat DAF deficient model may serve as a useful tool to study the role of this complement activation regulator in complement-dependent forms of kidney injury.

Disruption of the neuregulin 1 gene in the rat alters HPA axis activity and behavioral responses to environmental stimuli.

Exposure to stress can result in an increased risk for psychiatric disorders, especially among genetically predisposed individuals. Neuregulin 1 (NRG1) is a susceptibility gene for schizophrenia and is also associated with psychotic bipolar disorder. In the rat, the neurons of the hypothalamic paraventricular nucleus show strong expression of Nrg1 mRNA. In patients with schizophrenia, a single nucleotide polymorphism in the 5' region of NRG1 interacts with psychosocial stress to affect reactivity to expressed emotion. However, there is virtually no information on the role of NRG1 in hypothalamic-pituitary-adrenal axis function, and whether the protein is expressed in the paraventricular nucleus is unknown. The present studies utilize a unique line of Nrg1 hypomorphic rats (Nrg1(Tn)) generated by gene trapping with the Sleeping Beauty transposon. We first established that the Nrg1(Tn) rats displayed reduced expression of both the mRNA and protein corresponding to the Type II NRG1 isoform. After confirming, using wild type animals, that Type II NRG1 is expressed in the neurocircuitry involved in regulating hypothalamic-pituitary-adrenal axis responses to environmental stimuli, the Nrg1(Tn) rats were then used to test the hypothesis that altered expression of Type II NRG1 disrupts stress regulation and reactivity. In support of this hypothesis, Nrg1(Tn) rats have disrupted basal and acute stress recovery corticosterone secretion, differential changes in expression of glucocorticoid receptors in the pituitary, paraventricular nucleus and hippocampus, and a failure to habituate to an open field. Together, these findings point to NRG1 as a potential novel regulator of neuroendocrine responses to stress as well as behavioral reactivity.

Continuous high thoracic epidural administration of morphine with bupivacaine after thoracotomy.

BACKGROUND AND OBJECTIVES: The purpose of the study is to determine the ideal concentration of morphine when given with bupivacaine as a continuous high thoracic epidural infusion for postthoracotomy pain. METHODS: In a prospective study, 60 patients scheduled for thoracic surgery received a high thoracic epidural catheter. Postoperative analgesia was provided by a continuous epidural infusion for 3 days. The patients were randomly divided into two groups: group 1 (loading dose 1 mg morphine epidurally and continuous infusion of bupivacaine 0.75% + 0.2 mg/mL morphine at an infusion rate of 0.8 mL/hr); group 2 (loading dose 0.5 mg morphine epidurally and continuous infusion of bupivacaine 0.75% + 0.1 mg/mL morphine at an infusion rate of 0.8 mL/hr). RESULTS: The visual analog scales were not different at rest but with exercise in group 1 there was better pain relief than in group 2. The number of patients requiring supplementation of analgesia in group 2 (n = 42) was six times that of group 1 (n = 7). PaCO2 increased in both groups during the first postoperative day. There was no difference in the incidence of side effects between the two groups. CONCLUSIONS: Continuous high thoracic epidural administration 0.2 mg/mL morphine in bupivacaine 0.75% at an infusion rate of 0.8 mL/hr with a loading dose of 1 mg morphine is an effective dose for postthoracotomy pain relief in rest, and more important, during exercise.