Xueping Ee

Lactobacillus probiotic protects intestinal epithelium from radiation injury in a TLR-2/cyclo-oxygenase-2-dependent manner

Background The small intestinal epithelium is highly sensitive to radiation and is a major site of injury during radiation therapy and environmental overexposure. Objective To examine probiotic bacteria as potential radioprotective agents in the intestine. Methods 8-week-old C57BL/6 wild-type or knockout mice were administered probiotic by gavage for 3 days before 12 Gy whole body radiation. The intestine was evaluated for cell-positional apoptosis (6 h) and crypt survival (84 h). Results Gavage of 5×107 Lactobacillus rhamnosus GG (LGG) improved crypt survival about twofold (p<0.01); the effect was observed when administered before, but not after, radiation. Conditioned medium (CM) from LGG improved crypt survival (1.95-fold, p<0.01), and both LGG and LGG-CM reduced epithelial apoptosis particularly at the crypt base (33% to 18%, p<0.01). LGG was detected in the distal ileal contents after the gavage cycle, but did not lead to a detectable shift in bacterial family composition. The reduction in epithelial apoptosis and improved crypt survival offered by LGG was lost in MyD88−/−, TLR-2−/− and cyclo-oxygenase-2−/− (COX-2) mice but not TLR-4−/− mice. LGG administration did not lead to increased jejunal COX-2 mRNA or prostaglandin E2 levels or a change in number of COX-2-expressing cells. However, a location shift was observed in constitutively COX-2-expressing cells of the lamina propria from the villi to a position near the crypt base (villi to crypt ratio 80:20 for control and 62:38 for LGG; p<0.001). Co-staining revealed these COX-2-expressing small intestinal lamina propria cells to be mesenchymal stem cells. Conclusions LGG or its CM reduce radiation-induced epithelial injury and improve crypt survival. A TLR-2/MyD88 signalling mechanism leading to repositioning of constitutive COX-2-expressing mesenchymal stem cells to the crypt base is invoked.

Hyaluronic acid regulates normal intestinal and colonic growth in mice

Hyaluronic acid (HA), a component of the extracellular matrix, affects gastrointestinal epithelial proliferation in injury models, but its role in normal growth is unknown. We sought to determine the effects of exogenous HA on intestinal and colonic growth by intraperitoneal injection of HA twice a week into C57BL/6 mice from 3 to 8 wk of age. Similarly, to determine the effects of endogenous HA on intestinal and colonic growth, we administered PEP-1, a peptide that blocks the binding of HA to its receptors, on the same schedule. In mice treated with exogenous HA, villus height and crypt depth in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were increased. In mice treated with PEP-1, intestinal and colonic length were markedly decreased and crypt depth and villus height in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were decreased. Administration of HA was associated with increased levels of EGF (intestine) and IGF-I (colon), whereas administration of PEP-1 was associated with decreased levels of IGF-I (intestine) and epiregulin (colon). Exogenous HA increases intestinal and colonic epithelial proliferation, resulting in hyperplasia. Blocking the binding of endogenous HA to its receptors results in decreased intestinal and colonic length and a mucosal picture of hypoplasia, suggesting that endogenous HA contributes to the regulation of normal intestinal and colonic growth.

Imaging of radicals following injury or acute stress in peripheral nerves with activatable fluorescent probes

Peripheral nerve injury evokes a complex cascade of chemical reactions including generation of molecular radicals. Conversely, the reactions within nerve induced by stress are difficult to directly detect or measure to establish causality. Monitoring these reactions in vivo would enable deeper understanding of the nature of the injury and healing processes. Here, we utilized near-infrared fluorescence molecular probes delivered via intra-neural injection technique to enable live, in vivo imaging of tissue response associated with nerve injury and stress. These initially quenched fluorescent probes featured specific sensitivity to hydroxyl radicals and become fluorescent upon encountering reactive oxygen species (ROS). Intraneurally delivered probes demonstrated rapid activation in injured rat sciatic nerve but minimal activation in normal, uninjured nerve. In addition, these probes reported activation within sciatic nerves of living rats after a stress caused by a pinprick stimulus to the abdomen. This imaging approach was more sensitive to detecting changes within nerves due to the induced stress than other techniques to evaluate cellular and molecular changes. Specifically, neither histological analysis of the sciatic nerves, nor the expression of pain and stress associated genes in dorsal root ganglia could provide statistically significant differences between the control and stressed groups. Overall, the results demonstrate a novel imaging approach to measure ROS in addition to the impact of ROS within nerve in live animals.

The Effect of Short Nerve Grafts in Series on Axonal Regeneration Across Isografts or Acellular Nerve Allografts.

PURPOSE To evaluate the regenerative effect of the additional suture line when using either isografts (ISOs) or acellular nerve allografts (ANAs) placed end-to-end to span a short gap in a rat model. METHODS Rat sciatic nerves were transected and repaired with 2-cm nerve grafts (ISO or ANA). The grafts were 2 cm in length or a 1-cm segment was connected end-to-end to a 1-cm segment to yield a 2-cm length. At 8 weeks, extensor digitorum longus (EDL) muscle force and mass were measured. Nerves were harvested for histomorphometry. In a separate parallel study, the nerves were harvested 2 weeks following graft implantation to assess gene expression changes. RESULTS All grafts demonstrated regeneration across the 2-cm segment(s). The additional suture line did not result in statistical differences in the number of myelinated nerve fibers that reached the distal nerve. However, when the graft types were compared, there was a significant decrease in nerve fibers in the ANA groups. The EDL muscle mass was significantly greater by using nerve ISOs compared with ANAs, regardless of an additional suture line, but there were no statistical differences noted in EDL muscle force. Gene expression analysis did not differ owing to an additional suture line. CONCLUSIONS Minimal axonal loss and no functional deficits were identified with an additional suture line in this rodent short nerve gap model. CLINICAL RELEVANCE Placing nerve grafts in series is a viable option for treating short nerve gaps; however, the use of autografts remains preferable over the use of ANAs.

Nerve stepping stone has minimal impact in aiding regeneration across long acellular nerve allografts

Introduction: Acellular nerve allografts (ANAs) yield less consistent favorable outcomes compared with autografts for long gap reconstructions. We evaluated whether a hybrid ANA can improve 6‐cm gap reconstruction. Methods: Rat sciatic nerve was transected and repaired with either 6‐cm hybrid or control ANAs. Hybrid ANAs were generated using a 1‐cm cellular isograft between 2.5‐cm ANAs, whereas control ANAs had no isograft. Outcomes were assessed by graft gene and marker expression (n = 4; at 4 weeks) and motor recovery and nerve histology (n = 10; at 20 weeks). Results: Hybrid ANAs modified graft gene and marker expression and promoted modest axon regeneration across the 6‐cm defect compared with control ANA (P < 0.05), but yielded no muscle recovery. Control ANAs had no appreciable axon regeneration across the 6‐cm defect. Discussion: A hybrid ANA confers minimal motor recovery benefits for regeneration across long gaps. Clinically, the authors will continue to reconstruct long nerve gaps with autografts. Muscle Nerve 57: 260–267, 2018

Transgenic SCs expressing GDNF-IRES-DsRed impair nerve regeneration within acellular nerve allografts†

Providing temporally regulated glial cell line‐derived neurotrophic factor (GDNF) to injured nerve can promote robust axon regeneration. However, it is poorly understood why providing highly elevated levels of GDNF to nerve can lead to axon entrapment in the zone containing elevated GDNF. This limited understanding represents an obstacle to the translation of GDNF therapies to treat nerve injuries clinically. Here, we investigated how transgenic Schwann cells (SCs) overexpressing GDNF‐IRES‐DsRed impact nerve regeneration. Cultured primary SCs were transduced with lentiviruses (GDNF‐overexpressing transgenic SCs), one of which provides the capability to express high levels of GDNF and regulate temporal GDNF expression. These SC groups were transplanted into acellular nerve allografts (ANAs) bridging a 14 mm rat sciatic nerve defect. GDNF‐overexpressing transgenic SCs expressing GDNF for as little as 1 week decreased axon regeneration across ANAs and caused extensive extracellular matrix (ECM) remodeling. To determine whether additional gene expression changes beyond GDNF transgene expression occurred in GDNF‐overexpressing transgenic SCs, microarray analysis of GDNF‐overexpressing transgenic SCs compared to untreated SCs was performed. Microarray analysis revealed a set of common genes regulated in transgenic SC groups expressing high levels of GDNF compared to untreated SCs. A co‐culture model of GDNF‐overexpressing transgenic SCs with fibroblasts (FBs) revealed differential FB ECM‐related gene expression compared to untreated SCs. These data suggest a component of axon entrapment is independent of GDNFs impact on axons. Biotechnol. Bioeng. 2017;114: 2121–2130.

Lactobacillus rhamnosus GG protects the intestinal epithelium from radiation injury through release of lipoteichoic acid, macrophage activation and the migration of mesenchymal stem cells

Objective Lactobacillus rhamnosus GG (LGG), a probiotic, given by gavage is radioprotective of the mouse intestine. LGG-induced radioprotection is toll-like receptor 2 (TLR2) and cyclooxygenase-2 (COX-2)-dependent and is associated with the migration of COX-2+mesenchymal stem cells (MSCs) from the lamina propria of the villus to the lamina propria near the crypt epithelial stem cells. Our goals were to define the mechanism of LGG radioprotection including identification of the TLR2 agonist, and the mechanism of the MSC migration and to determine the safety and efficacy of this approach in models relevant to clinical radiation therapy. Design Intestinal radioprotection was modelled in vitro with cell lines and enteroids as well as in vivo by assaying clinical outcomes and crypt survival. Fractionated abdominal and single dose radiation were used along with syngeneic CT26 colon tumour grafts to assess tumour radioprotection. Results LGG with a mutation in the processing of lipoteichoic acid (LTA), a TLR2 agonist, was not radioprotective, while LTA agonist and native LGG were. An agonist of CXCR4 blocked LGG-induced MSC migration and LGG-induced radioprotection. LGG given by gavage induced expression of CXCL12, a CXCR4 agonist, in pericryptal macrophages and depletion of macrophages by clodronate liposomes blocked LGG-induced MSC migration and radioprotection. LTA effectively protected the normal intestinal crypt, but not tumours in fractionated radiation regimens. Conclusions LGG acts as a ‘time-release capsule’ releasing radioprotective LTA. LTA then primes the epithelial stem cell niche to protect epithelial stem cells by triggering a multicellular, adaptive immune signalling cascade involving macrophages and PGE2 secreting MSCs. Trial registration number NCT01790035; Pre-results.

Abstract 53: Discrepancies in Senescence and Protein Expression from Cells in Nerve Autografts Compared to Injured Nerve

RESULTS: Axon counts clearly demonstrated disproportionately reduced regeneration in the longer graft compared to the short graft when measuring at equivalent distances to the spinal cord: the number of regenerated axons in the long graft was 57% of those regenerated in the short graft. Additionally, retrograde labeling showed significantly fewer motoneurons were found to be regenerating axons to long grafts compared to the short graft repairs. Cell composition amongst the different grafts and injured nerve were surprisingly consistent: groups had similar total numbers of cells and similar proportions of fibroblasts, Schwann cells, and macrophages. Immunohistochemical analysis showed an increased percentage of cells with senescent markers in the long grafts and in the “normal” nerve proximal to the graft. While gene expression for senescent markers was increased in both long and short nerve grafts, these markers remained elevated over time in the long grafts. Interestingly, GDNF and IL-6 expression was elevated in the long grafts compared to the short. Finally, immunohistochemistry revealed increased NOTCH signalling in the long grafts. CONCLUSION: Comparison of short and long grafts, at points equidistant to the spinal cord, showed reduced axon regeneration and reduced number of motoneurons regenerating axons. Given this reduced regeneration in the presence of a long graft and the finding that cellular composition was unchanged, immunohistochemistry and gene expression were used to identify gene and protein expression differences in the grafts.The longer grafts were found to have a greater proportion of senescent cells and the nerve proximal to the graft even showed senescent changes. Gene expression associated with senescent was increased in the grafts but retained over time in the longer grafts. Prior work has linked increased NOTCH signalling to reduced neurite extension from dorsal root ganglia neurons and the current work links increasing graft length with increased NOTCH signalling. These protein and gene expression changes give insight as to the poor clinical outcomes associated with autografts and provide targets for improvement.