Paneth cells are the primary source of C-type lysozyme, a b-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn's disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1 À/À hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti-and pro-inflammatory responses, with implications for IBD.

Intestinal epithelial Na+/H+ exchange facilitated by the apical NHE3 (Slc9a3) is a highly-regulated process inhibited by intestinal pathogens and in inflammatory bowel diseases. NHE3−/− mice develop spontaneous, bacterially-mediated colitis, and IBD-like dysbiosis. Disruption of epithelial Na+/H+ exchange in IBD may thus represent a host response contributing to the altered gut microbial ecology, and may play a pivotal role in modulating the severity of inflammation in a microbiome-dependent manner. To test whether microbiome fostered in an NHE3-deficient environment is able to drive mucosal immune responses affecting the onset or severity of colitis, we performed a series of cohousing experiments and fecal microbiome transplants into germ-free Rag-deficient or IL-10−/− mice. We determined that in the settings where the microbiome of NHE3-deficient mice was stably engrafted in the recipient host, it was able accelerate the onset and amplify severity of experimental colitis. NHE3-deficiency was characterized by the reduction in pH-sensitive butyrate-producing Firmicutes families Lachnospiraceae and Ruminococcaceae (Clostridia clusters IV and XIVa), with an expansion of inflammation-associated Bacteroidaceae. We conclude that the microbiome fostered by impaired epithelial Na+/H+ exchange enhances the onset and severity of colitis through disruption of the gut microbial ecology.

Buprenorphine is a commonly used opioid for mitigating pain in laboratory mice after surgical procedures; however, the dosing interval necessary for standard buprenorphine may require treatment every 4 to 6 h to maintain an adequate plane of analgesia. An alternative formulation that provides prolonged plasma concentration with long-lasting effects would be beneficial in achieving steady-state analgesia. We evaluated a long-lasting and highly concentrated formulation of buprenorphine(Bup-LHC) in mice. Pharmacokinetic analysis was performed to assess plasma concentrations in male C57BL/6J (B6)and female CD1 mice after subcutaneous injection of 0.9 mg/kg. The Bup-LHC formulation provided plasma drug levels that exceeded the therapeutic level for at least 12 h in male B6 mice and was below therapeutic levels by 8 h in female CD1 mice. An experimental laparotomy model was used to assess analgesic efficacy. Female CD1 mice were treated with either Bup-LHC (0.9 mg/kg) or saline at 1 h before undergoing an ovariectomy via a ventral laparotomy. At 3, 6, 12, 24, and 48 h after surgery, pain was assessed based on the following behaviors: orbital tightness, grooming, wound licking, rearing, arched posture, ataxia, piloerection, nest building, and general activity. At 3 and 6 h after surgery, Bup-LHC–treated mice had significantly less wound licking and orbital tightness and considerably higher activity levels than did saline-treated mice. At 12 h, wound licking, orbital tightness and activity in Bup-LHC–treated mice were no longer significantly different from those of saline-treated mice. The results of this study suggest that Bup-LHC at 0.9 mg/kg provides sufficient plasma concentrations for analgesia in mice for 6 to 12 h after administration, as demonstrated behaviorally for at least 6 h after surgery.

Long-term in vivo imaging in adult zebrafish (i.e., 1-24 h) has been limited by the fact that regimens for long-term anesthesia in embryos and larvae are ineffective in adults. Here, we examined the potential for dynamic administration of benzocaine to enable long-term anesthesia in adult zebrafish. We developed a computer-controlled perfusion system comprised of programmable peristaltic pumps that enabled automatic exchange between anesthetic and system water. Continuous administration of benzocaine in adult zebrafish resulted in a mean time to respiratory arrest of 5.0 h and 8-h survival of 14.3%. We measured characteristic sedation and recovery times in response to benzocaine, and used them to devise an intermittent dosing regimen consisting of 14.5 min of benzocaine followed by 5.5 min of system water. Intermittent benzocaine administration in adult zebrafish resulted in a mean time to respiratory arrest of 7.6 h and 8-h survival of 71.4%. Finally, we performed a single 24-h trial and found that intermittent dosing maintained anesthesia in an adult zebrafish over the entire 24-h period. In summary, our studies demonstrate the potential for dynamic administration of benzocaine to enable prolonged anesthesia in adult zebrafish, expanding the potential for imaging in adult physiologies that unfold over 1-24 h.

No abstract