Position statement by the Pelvic Floor Society on behalf of the Association of Coloproctology of Great Britain and Ireland on the use of mesh in ventral mesh rectopexy.

The following position statement forms part of a response to the current concerns regarding use of mesh to perform rectal prolapse surgery. It highlights the actions being pursued by the Pelvic Floor Society (TPFS) regarding clinical governance in relation to ventral mesh rectopexy (VMR). The following are summary recommendations. Available evidence suggests that mesh morbidity for VMR is far lower than that seen in transvaginal procedures (the main subject of current concern) and lower than that observed following other abdomino-pelvic procedures for urogenital prolapse, e.g. laparoscopic sacrocolpopexy. VMR should be performed by adequately trained surgeons who work within a multidisciplinary team (MDT) framework. Within this, it is mandatory to discuss all patients considered for surgery at an MDT meeting. Clinical outcomes of surgery and any complications resulting from surgery should be recorded in the TPFS-hosted national database (registry) available for this purpose; in addition, all patients should be considered for entry into ongoing and planned UK/European randomized studies where this is feasible. A move towards accreditation of UK units performing VMR will improve performance and outcomes in the long term. An enhanced programme of training including staged porcine, cadaveric and preceptorship sessions will ensure the competence of surgeons undertaking VMR. Enhanced consent forms and patient information booklets are being developed, and these will help both surgeons and patients. There is weak observational evidence that technical aspects of the procedure can be optimized to reduce morbidity rates. Suture material choice may contribute towards morbidity. The available evidence is insufficient to support the use of one mesh over another (biologic vs synthetic); however, the use of polyester mesh is associated with increased morbidity.

Protective effect of hormone therapy among women with hysterectomy/oophorectomy.

Study question: Does exposure to menopausal hormone therapy (MHT) in mid-aged women alter their risk of cardiovascular disease (CVD) mortality and all-cause mortality? Summary answer: MHT soon after menopause is unlikely to increase the risk of CVD mortality or all-cause mortality and may have a protective effect for women with hysterectomy/oophorectomy. What is known already: The balance of benefits and risks of MHT are currently unclear and may differ according to when treatment starts and whether women have an intact uterus. Study design size, duration: A total of 13 715 participants from the mid-aged population-based cohort (born 1946-1951) of the Australian Longitudinal Study on Women's Health (ALSWH) were followed from 1998 to 2013. Participants/materials setting methods: The measures included cardiovascular and all-cause mortality, exposure to MHT and menopausal status (based on 3-yearly self-reports). Electronic prescriptions data on MHT were also available from mid-2002 onwards. At each follow-up survey wave, participants were classified as: an existing user of MHT, an initiator of MHT or a non-initiator of MHT. Main results and the role of chance: After adjusting for confounding variables, existing users of MHT had a reduced risk (hazard ratio 0.63; 95% CI, 0.43-0.92) of CVD mortality compared with non-initiators. Insufficient evidence of an association was identified for initiators of MHT (0.66; 0.35-1.24). For all-cause mortality, risks were reduced for both initiators (0.69; 0.55-0.87) and existing users (0.80; 0.70-0.91). In a subgroup analysis, women with hysterectomy/oophorectomy had lower risks of CVD mortality for both initiators (0.14; 0.02-0.98) and existing users (0.55; 0.34-0.90), but no evidence of an association was found for women whose MHT commenced during or after menopause. Similarly for all-cause mortality, only the women with hysterectomy/oophorectomy had lower risks for both initiators (0.47; 0.31-0.70) and existing users (0.69; 0.58-0.82). Limitations, reasons for caution: Limitations include the observational nature of the study, the small number of deaths, MHT use being self-reported and the classification of menopausal status also being based on self-reported information. Wider implications of the findings: Women considering MHT soon after menopause can be reassured that the treatment is unlikely to increase their risk of CVD mortality or all-cause mortality. Study funding/competing interest(s): The Australian Longitudinal Study on Women's Health is funded by the Australian Department of Health. G.D.M. is funded by the Australian Research Council Future Fellowship. L.C. was funded by a China scholarship council (CSC) graduate scholarship. All authors report no conflict of interest. Trial registration number: N/A.

Three-dimensional microstructure characterisation of thermoplastic polyolefin blends.

The size, shape and distribution of different phases in thermoplastic polyolefin (TPO) blends and composites are critical to the properties of the materials, but can be difficult to characterise. Here we report the combination of heavy metal staining and focused ion beam - scanning electron microscopy (FIB-SEM) to reveal the three-dimensional (3D) structure of an elastomer-modified poly(propylene) and a talc filled elastomer-modified poly(propylene). High-quality, high-resolution serial images were collected and the 3D structures were characterised quantitatively.

The genomic architecture of resistance to Campylobacter jejuni intestinal colonisation in chickens.

BACKGROUND: Campylobacter is the leading cause of foodborne diarrhoeal illness in humans and is mostly acquired from consumption or handling of contaminated poultry meat. In the absence of effective licensed vaccines and inhibitors, selection for chickens with increased resistance to Campylobacter could potentially reduce its subsequent entry into the food chain. Campylobacter intestinal colonisation levels are influenced by the host genetics of the chicken. In the present study, two chicken populations were used to investigate the genetic architecture of avian resistance to colonisation: (i) a back-cross of two White Leghorn derived inbred lines [(61 x N) x N] known to differ in resistance to Campylobacter colonisation and (ii) a 9(th) generation advanced intercross (61 x N) line. RESULTS: The level of colonisation with Campylobacter jejuni following experimental infection was found to be a quantitative trait. A back-cross experiment using 1,243 fully informative single nucleotide polymorphism (SNP) markers revealed quantitative trait loci (QTL) on chromosomes 7, 11 and 14. In the advanced intercross line study, the location of the QTL on chromosome 14 was confirmed and refined and two new QTLs were identified located on chromosomes 4 and 16. Pathway and re-sequencing data analysis of the genes located in the QTL candidate regions identified potential pathways, networks and candidate resistance genes. Finally, gene expression analyses were performed for some of the candidate resistance genes to support the results. CONCLUSION: Campylobacter resistance in chickens is a complex trait, possibly involving the Major Histocompatibility Complex, innate and adaptive immune responses, cadherins and other factors. Two of the QTLs for Campylobacter resistance are co-located with Salmonella resistance loci, indicating that it may be possible to breed simultaneously for enhanced resistance to both zoonoses.

The long view: Salmonella--the last forty years.

As a part of the 40th anniversary celebrations of Avian Pathology we review the last four decades of Salmonella research which has led to major progress in our understanding of the bacteriology and infection biology of the organism through the huge advances in molecular biology and immunology that have accompanied technical advances in biology generally. In many countries combinations of improvements in management, sometimes under legislative pressure and supported by a number of basic biological interventions, have resulted in reductions in incidence in the Salmonella serovars that are commonly associated with food-poisoning to unprecedented low levels in parent flocks, broilers and layers. Utilisation of the information generated during the past few decades should improve the efficacy of surveillance and biological interventions both for the intestinal carriage that is associated most frequently with human infection and also for systemic diseases, including fowl typhoid and pullorum disease. These two diseases continue to be major economic problems in many countries where the possibilities for improvements in hygiene may be limited but which, nevertheless, are increasingly a significant part of the global economy in poultry meat.

GSTM1 null and NAT2 slow acetylation genotypes, smoking intensity and bladder cancer risk: results from the New England bladder cancer study and NAT2 meta-analysis.

Associations between bladder cancer risk and NAT2 and GSTM1 polymorphisms have emerged as some of the most consistent findings in the genetic epidemiology of common metabolic polymorphisms and cancer, but their interaction with tobacco use, intensity and duration remain unclear. In a New England population-based case-control study of urothelial carcinoma, we collected mouthwash samples from 1088 of 1171 cases (92.9%) and 1282 of 1418 controls (91.2%) for genotype analysis of GSTM1, GSTT1 and NAT2 polymorphisms. Odds ratios and 95% confidence intervals of bladder cancer among New England Bladder Cancer Study subjects with one or two inactive GSTM1 alleles (i.e. the 'null' genotype) were 1.26 (0.85-1.88) and 1.54 (1.05-2.25), respectively (P-trend = 0.008), compared with those with two active copies. GSTT1 inactive alleles were not associated with risk. NAT2 slow acetylation status was not associated with risk among never (1.04; 0.71-1.51), former (0.95; 0.75-1.20) or current smokers (1.33; 0.91-1.95); however, a relationship emerged when smoking intensity was evaluated. Among slow acetylators who ever smoked at least 40 cigarettes/day, risk was elevated among ever (1.82; 1.14-2.91, P-interaction = 0.07) and current heavy smokers (3.16; 1.22-8.19, P-interaction = 0.03) compared with rapid acetylators in each category; but was not observed at lower intensities. In contrast, the effect of GSTM1-null genotype was not greater among smokers, regardless of intensity. Meta-analysis of the NAT2 associations with bladder cancer showed a highly significant relationship. Findings from this large USA population-based study provided evidence that the NAT2 slow acetylation genotype interacts with tobacco smoking as a function of exposure intensity.

Salmonella enterica serovar typhimurium colonizing the lumen of the chicken intestine grows slowly and upregulates a unique set of virulence and metabolism genes.

The pattern of global gene expression in Salmonella enterica serovar Typhimurium bacteria harvested from the chicken intestinal lumen (cecum) was compared with that of a late-log-phase LB broth culture using a whole-genome microarray. Levels of transcription, translation, and cell division in vivo were lower than those in vitro. S. Typhimurium appeared to be using carbon sources, such as propionate, 1,2-propanediol, and ethanolamine, in addition to melibiose and ascorbate, the latter possibly transformed to d-xylulose. Amino acid starvation appeared to be a factor during colonization. Bacteria in the lumen were non- or weakly motile and nonchemotactic but showed upregulation of a number of fimbrial and Salmonella pathogenicity island 3 (SPI-3) and 5 genes, suggesting a close physical association with the host during colonization. S. Typhimurium bacteria harvested from the cecal mucosa showed an expression profile similar to that of bacteria from the intestinal lumen, except that levels of transcription, translation, and cell division were higher and glucose may also have been used as a carbon source.

Genome-wide SNP analysis identifies major QTL for Salmonella colonization in the chicken.

Salmonella-infected poultry products are a major source of human Salmonella infection. The prophylactic use of antimicrobials in poultry production was recently banned in the EU, increasing the need for alternative methods to control Salmonella infections in poultry flocks. Genetic selection of chickens more resistant to Salmonella colonization provides an attractive means of sustainably controlling the pathogen in commercial poultry flocks and its subsequent entry into the food chain. Analysis of different inbred chickens has shown that individual lines are consistently either susceptible or resistant to the many serovars of Salmonella that have been tested. In this study, two inbred chicken lines with differential susceptibility to Salmonella colonization (61 ((R)) and N((S)) ) were used in a backcross experimental design. Unlike previous studies that used a candidate gene approach or low-density genome-wide screens, we have exploited a high-density marker set of 1255 SNPs covering the whole genome to identify quantitative trait loci (QTL). Analysis of log-transformed caecal bacterial levels between the parental lines revealed a significant difference at 1, 2, 3 and 4 days post-infection (P < 0.05). Analysis of the genotypes of the backcross (F1 × N) population (n = 288) revealed four QTL on chromosomes 2, 3, 12 and 25 for the two traits examined in this study: log-transformed bacterial counts in the caeca and presence of a hardened caseous caecal core. These included one genome-wide significant QTL on chromosome 2 at 20 Mb and three additional QTL, on chromosomes 3, 12 and 25 at 96, 15 and 1 Mb, respectively, which were significant at the chromosome-wide level (P < 0.05). The results generated in this study will inform future breeding strategies to control these pathogens in commercial poultry flocks.

Improved hospital-level risk adjustment for surveillance of healthcare-associated bloodstream infections: a retrospective cohort study.

BACKGROUND: To allow direct comparison of bloodstream infection (BSI) rates between hospitals for performance measurement, observed rates need to be risk adjusted according to the types of patients cared for by the hospital. However, attribute data on all individual patients are often unavailable and hospital-level risk adjustment needs to be done using indirect indicator variables of patient case mix, such as hospital level. We aimed to identify medical services associated with high or low BSI rates, and to evaluate the services provided by the hospital as indicators that can be used for more objective hospital-level risk adjustment. METHODS: From February 2001-December 2007, 1719 monthly BSI counts were available from 18 hospitals in Queensland, Australia. BSI outcomes were stratified into four groups: overall BSI (OBSI), Staphylococcus aureus BSI (STAPH), intravascular device-related S. aureus BSI (IVD-STAPH) and methicillin-resistant S. aureus BSI (MRSA). Twelve services were considered as candidate risk-adjustment variables. For OBSI, STAPH and IVD-STAPH, we developed generalized estimating equation Poisson regression models that accounted for autocorrelation in longitudinal counts. Due to a lack of autocorrelation, a standard logistic regression model was specified for MRSA. RESULTS: Four risk services were identified for OBSI: AIDS (IRR 2.14, 95% CI 1.20 to 3.82), infectious diseases (IRR 2.72, 95% CI 1.97 to 3.76), oncology (IRR 1.60, 95% CI 1.29 to 1.98) and bone marrow transplants (IRR 1.52, 95% CI 1.14 to 2.03). Four protective services were also found. A similar but smaller group of risk and protective services were found for the other outcomes. Acceptable agreement between observed and fitted values was found for the OBSI and STAPH models but not for the IVD-STAPH and MRSA models. However, the IVD-STAPH and MRSA models successfully discriminated between hospitals with higher and lower BSI rates. CONCLUSION: The high model goodness-of-fit and the higher frequency of OBSI and STAPH outcomes indicated that hospital-specific risk adjustment based on medical services provided would be useful for these outcomes in Queensland. The low frequency of IVD-STAPH and MRSA outcomes indicated that development of a hospital-level risk score was a more valid method of risk adjustment for these outcomes.

Effect of an acute necrotic bacterial gill infection and feed deprivation on the metabolic rate of Atlantic salmon Salmo salar.

In this study, experiments were conducted to examine the effect of an acute necrotic bacterial gill infection on the metabolic rate (M(O2)) of Atlantic salmon Salmo salar. Fed and unfed Atlantic salmon smolts were exposed to a high concentration (5 x 10(12) CFU ml(-1)) of the bacteria Tenacibaculum maritimum, their routine and maximum metabolic rates (M(O2rout) and M(O2max), respectively) were measured, and relative metabolic scope determined. A significant decrease in metabolic scope was found for both fed and unfed infected groups. Fed infected fish had a mean +/- standard error of the mean (SEM) decrease of 2.21 +/- 0.97 microM O2 g(-1) h(-1), whilst unfed fish a mean +/- SEM decrease of 3.16 +/- 1.29 microM O2 g(-1) h(-1). The decrease in metabolic scope was a result of significantly increased M(O2rout) of both fed and unfed infected salmon. Fed infected fish had a mean +/- SEM increase in M(O2rout) of 1.86 +/- 0.66 microM O2 g(-1) h(-1), whilst unfed infected fish had a mean +/- SEM increase of 2.16 +/- 0.72 microM O2 g(-1) h(-1). Interestingly, all groups maintained M(O2max) regardless of infection status. Increases in M(O2rout) corresponded to a significant increase in blood plasma osmolality. A decrease in metabolic scope has implications for how individuals allocate energy; fish with smaller metabolic scope will have less energy to allocate to functions such as growth, reproduction and immune response, which may adversely affect the efficiency of fish growth.

Development of a large-animal human brain tumor xenograft model in immunosuppressed cats.

A large-animal model was developed to facilitate the noninvasive investigation of the effect on the human glioma-derived D-54 MG (glioblastoma multiforme) continuous cell line of a variety of therapeutic regimens. Twenty random-bred male cats were inoculated intracerebrally with 1 x 10(7) D-54 MG tumor cells after being initiated on one of three preparatory regimens of cyclosporin A p.o. Reproducible success of D-54 MG xenotransplantation (100%, 6 of 6 cats) was achieved only after pretreatment with 120 mg cyclosporin A p.o. (24-30 mg/kg) daily for greater than or equal to 10 days prior to tumor implantation. High-performance liquid chromatography-derived whole blood cyclosporin A 12-h trough levels of greater than or equal to 640 ng/ml were seen in successful implants. Lesions ranging from 2 to 20 mm in diameter were seen in cats sacrificed 27-44 days after implantation with no growth seen in control animals. Histopathological examination revealed the tumors to be well-circumscribed anaplastic intracerebral tumors with some invasion into surrounding host parenchyma. Perivascular lymphocytic cuffing was observed, but intratumoral lymphocytic infiltration was minimal. Gadolinium-EDTA-enhanced nuclear magnetic resonance imaging provided accurate tumor localization in T1-weighted images (TE 26 ms; TR 600 ms). Biochemical tests of kidney, liver, and hematological function were within normal limits, although 10% (2 of 20) of the animals developed gingival hyperplasia, and 5% (1 of 20) developed intussusception. The reproducible growth of the D-54 MG human glioblastoma cell line in a large-animal model eliminates many of the limitations associated with the standard nude mouse/rat model, thereby providing a novel test bed for a variety of imaging modalities as well as for drug immunoconjugate localization and toxicity studies.

FMS (CSF-1 receptor) and CSF-1 transcripts and protein are expressed by human breast carcinomas in vivo and in vitro.

The expression in vivo of FMS transcripts and antigen by neoplastic epithelial cells was demonstrated immunohistochemically or by in situ hybridization in sixteen of seventeen human breast carcinoma specimens and one case of sclerosing adenosis. Expression of CSF-1 receptor (FMS) transcripts and protein was also observed in vitro in two or three breast carcinoma-derived cell lines and was dramatically increased by dexamethasone, a potent glucocorticoid and inducer of mammary epithelial cell differentiation. Immunohistochemical staining with an anti-CSF-1 antibody identified neoplastic epithelial cell co-expression of fms and CSF-1 antigens in more than one-third of the fms-positive invasive carcinoma specimens. These results suggest that autocrine and paracrine interactions of the lymphohematopoietic cytokine CSF-1 and its receptor may participate in the biology of human mammary neoplasms.

Regulation of early peritoneal neutrophil migration by macrophage inflammatory protein-2 and mast cells in experimental peritonitis.

Neutrophil (PMN) migration into the peritoneal cavity in response to fecal peritonitis is an important mechanism of host defense against bacterial invasion. We show that the murine C-X-C (PMN-specific) chemokine, macrophage inflammatory protein-2 (MIP-2), on intraperitoneal injection in mice, causes PMN migration into the peritoneum. MIP-2 mRNA and protein were expressed by peritoneal leukocytes after cecal ligation and puncture (CLP) in mice and neutralization of MIP-2 reduced peritoneal PMN migration. A prerequisite for neutrophil-endothelial adhesion and subsequent migration from the circulation is selectin-mediated rolling. Pretreatment of mice with an anti-P-selectin antibody before intraperitoneal injection of MIP-2 significantly reduced peritoneal PMN migration. However, there are no reports that a C-X-C chemokine can up-regulate endothelial selectins. We postulated that MIP-2, when injected intraperitoneally, interacts with a cell that is known to release factors that up-regulate endothelial selectins. A likely candidate is the mast cell, which contains histamine and tumor necrosis factor alpha (TNF-alpha), and both of these factors induce selectins. Intraperitoneally injected MIP-2 caused an early significant increase in peritoneal TNF-alpha, whereas histamine levels were unaffected. In a subsequent experiment, mast cell-deficient mice and their normal controls were then injected intraperitoneally with MIP-2 or underwent CLP. Significantly fewer PMNs migrated into the peritoneal cavity in the mast cell-deficient mice after MIP-2 injection or CLP. Thus, our findings indicate that mast cells and MIP-2 are necessary for PMN migration into the peritoneum in response to intra-abdominal infection, and that MIP-2 appears to facilitate this through an increase in TNF-alpha release.

Histological evidence for the role of mechanical stress in modulating thermal denaturation of collagen.

The hyperthermia and thermal denaturation literatures reveal a time-temperature equivalency when heating cells or connective tissues: thermal damage increases with increasing temperature (for the same duration) and increases with increasing duration (for the same temperature). Recent findings conversely suggest that increasing the mechanical loading on a tissue during heating decreases the thermal damage (for a given temperature and duration of heating). Surprisingly, however, there are few histological correlates of such damage. In this paper, we show that progressive light microscopic changes - swelling of collagen bands, thickening of collagen-rich layers, hyalinization, and loss of birefringence approximately - correlate very well with both increased heating times and decreased mechanical loading. Increased mechanical stress is thus thermally protective and should be considered in the design of clinical procedures that use heating to treat diseases or injuries.

Biological risk indicators for recurrent non-specific low back pain in adolescents.

OBJECTIVES: A matched case-control study was carried out to evaluate biological risk indicators for recurrent non-specific low back pain in adolescents. METHODS: Adolescents with recurrent non-specific low back pain (symptomatic; n = 28; mean (SD) age 14.9 (0.7) years) and matched controls (asymptomatic; n = 28; age 14.9 (0.7) years) with no history of non-specific low back pain participated. Measures of stature, mass, sitting height, sexual maturity (Tanner self assessment), lateral flexion of the spine, lumbar sagittal plane mobility (modified Schober), hip range of motion (Leighton flexometer), back and hamstring flexibility (sit and reach), and trunk muscle endurance (number of sit ups) were performed using standardised procedures with established reliability. Backward stepwise logistic regression analysis was performed, with the presence/absence of recurrent low back pain as the dependent variable and the biological measures as the independent variables. RESULTS: Hip range of motion, trunk muscle endurance, lumbar sagittal plane mobility, and lateral flexion of the spine were identified as significant risk indicators of recurrent low back pain (p<0.05). Follow up analysis indicated that symptomatic subjects had significantly reduced lateral flexion of the spine, lumbar sagittal plane mobility, and trunk muscle endurance (p<0.05). CONCLUSIONS: Hip range of motion, abdominal muscle endurance, lumbar flexibility, and lateral flexion of the spine were risk indicators for recurrent non-specific low back pain in a group of adolescents. These risk indicators identify the potential for exercise as a primary or secondary prevention method.

Residual complement activity after incubation with myelin isolated from both the central and peripheral nervous tissues.

The relative ability of isolated central and peripheral nervous system myelin to interact with the complement system of plasma proteins was studied. The myelin used was a highly pure form, devoid of contamination by any subcellular organelles or membranes. Residual complement activity was a linear function of increasing quantities of myelin from 10 to 40 micrograms of myelin protein. Central and peripheral nervous system myelin showed identical residual complement activity at various temperatures above 7 degrees C and also after various time periods of incubation. The results show that central and peripheral nervous system myelin show equal ability to interact with complement, in spite of their different origin and differences in morphology and protein composition.

Rabbit blastocysts accumulate [3H]prostaglandins in vitro.

Rabbit blastocysts obtained on days 5, 6, and 6.8 of pregnancy were incubated in vitro in Tyrode's buffer with 3H-labeled prostaglandins (PGs). Accumulation of PGs was studied, using Whatman GF/F filters to separate bound and free ligands. The uptake and efflux of [3H]PGs were studied as a function of PG type, incubation time, temperature, and effect of metabolic inhibitors as well as age and number of blastocysts. Blastocysts of the same age accumulated approximately the same amount of [3H]PGE2 and [3H]PGF2 alpha from their environment; however, there was no apparent saturation over a PG concentration range of 1-1000 nM. Both the uptake and efflux of PG were age dependent, with older blastocysts accumulating more PGs. Approximately 90% of the [3H]PGs appear to be transported into the blastocoelic fluid, with little PG remaining in the blastomeres. PG accumulation was relatively insensitive to azide, ouabain, cyanide, or bromcresol green, but was affected by incubation at 0 C or the addition of indomethacin (10 micrograms/ml). No catabolism of the accumulated PGs was observed. The release of PGE2 in general did not differ from that of PGF2 alpha, except on day 6.8 of pregnancy when PGE2 was released more rapidly than on day 6. We conclude that rabbit blastocysts can accumulate PGs from their environment, which may imply a storage potential in the blastocyst and release before implantation.

Storage in vivo of [3H]prostaglandins by rabbit blastocysts.

Day 6 rabbit blastocysts that had previously been incubated with 10 nM [3H]prostaglandin E2 (PGE2), [3H] PGF2 alpha, or 0.5 microCi [3H]water were surgically transferred to the uteri of day 6 pseudopregnant recipient rabbits. At 1, 3, and 20 h after transfer, blastocysts were collected from the recipient rabbits and evaluated for retention of [3H]ligands. The transferred day 6 blastocysts were able to retain a significant proportion of the initial PGs for up to 20 h in vivo. These PGs were not metabolized while they remained in the blastocysts in vivo, as assessed by HPLC. The transferred blastocysts appeared normal and viable after 20 h in vivo, since implantation sites could be detected visually. These data support our hypothesis that blastocysts can sequester PGs from their environment in vivo and retain them unmetabolized during the critical period just before implantation.

Calcitonin gene-related peptide and corneal innervation: a developmental study in the rat.

The development of calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerves was studied in neonatal and adult rat corneas stained immunohistochemically according to an avidin biotin peroxidase procedure. At birth, rat corneas already contained dense plexuses of CGRP-LI nerve fibers. Most of the nerves entered the cornea in 12-15 prominent stromal nerve bundles located at regular intervals around the circumference of the cornea. Fibers in these bundles entered the epithelium approximately midway between the limbus and the center of the cornea and supplied extensive central and pericentral areas of the tissue. In addition, smaller numbers of axons entered the cornea individually and in small fascicles located in between the larger bundles and supplied mainly peripheral territory. In the epithelium, the CGRP-LI nerves formed a complex, highly anastomotic meshwork that ramified uniformly throughout central and peripheral areas of the tissues. Fibers in the plexus gave origin to numerous short, stout terminal axons that extended into the adjacent epithelium in all directions with no preferred orientation. During the first week of neonatal life, several changes in CGRP-LI innervation occurred: 1) the innervation density of the central and pericentral cornea increased relative to the peripheral cornea; 2) intraepithelial axons became progressively longer, increased in branching complexity, and oriented preferentially towards the center of the cornea; and 3) a dense innervation of the corneoscleral limbus and, in particular, the branches of the marginal artery, developed. Midway through the second week of life, immature versions of corneal epithelial "leashes," the dominant feature of the adult corneal innervation, were first observed. Over the next 10 days, the leash formations in the central and pericentral cornea gradually became more complex and gave rise to greater numbers of terminal axons, compared to developing leashes in the peripheral cornea. The mature pattern of corneal CGRP-LI innervation was reached on day 21 and remained constant (except for compensatory growth-related elongation of axons) for at least the first 6 months of life. Transection of the ophthalmomaxillary nerve or neonatal administration of the sensory neurotoxin capsaicin resulted in the total loss of CGRP-LI staining from the cornea. In contrast, removal of the superior cervical ganglion had no effect on corneal CGRP-LI staining. The extraordinary density and complexity of the CGRP-LI innervation of the rat cornea demonstrated at all stages of development in this study suggests that these nerves may play important roles in corneal sensory, reflex, and trophic functions.