Biofunctionalization of 3D-printed silicone implants with immunomodulatory hydrogels for controlling the innate immune response: An in vivo model of tracheal defect repair.

The recent advances in 3D-printed silicone (PDMS: polydimethylsiloxane) implants present prospects for personalized implants with highly accurate anatomical conformity. However, a potential adverse effect, such as granuloma formation due to immune reactions, still exists. One potential way to overcome this problem is to control the implant/host interface using immunomodulatory coatings. In this study, a new cytokine cocktail composed of interleukin-10 and prostaglandin-E2 was designed to decrease adverse immune reactions and promote tissue integration by fixing macrophages into M2 pro-healing phenotype for an extended period of time. In vitro, the cytokine cocktail maintained low levels of pro-inflammatory cytokine (TNF-α and IL-6) secretions and induced the secretion of IL-10 and the upregulation of multifunctional scavenging and sorting receptor stabilin-1, expressed by M2 macrophages. This cocktail was then loaded in a gelatine-based hydrogel to develop an immunomodulatory material that could be used as a coating for medical devices. The efficacy of this coating was demonstrated in an in vivo rat model during the reconstruction of a tracheal defect by 3D-printed silicone implants. The coating was stable on the silicone implants for over 2 weeks, and the controlled release of the cocktail components was achieved for at least 14 days. In vivo, only 33% of the animals with bare silicone implants survived, whereas 100% of the animals survived with the implant equipped with the immunomodulatory hydrogel. The presence of the hydrogel and the cytokine cocktail diminished the thickness of the inflammatory tissue, the intensity of both acute and chronic inflammation, the overall fibroblastic reaction, the presence of oedema and the formation of fibrinoid (assessed by histology) and led to a 100% survival rate. At the systemic level, the presence of immunomodulatory hydrogels significantly decreased pro-inflammatory cytokines such as TNF-α, IFN-γ, CXCL1 and MCP-1 levels at day 7 and significantly decreased IL-1α, IL-1ß, CXCL1 and MCP-1 levels at day 21. The ability of this new immunomodulatory hydrogel to control the level of inflammation once applied to a 3D-printed silicone implant has been demonstrated. Such thin coatings can be applied to any implants or scaffolds used in tissue engineering to diminish the initial immune response, improve the integration and functionality of these materials and decrease potential complications related to their presence.

Balanced Steady-State Free Precession Techniques Improve Detection of Residual Germ Cell Tumor for Treatment Planning.

BACKGROUND AND PURPOSE: Identification of a partial/complete chemotherapy response in pediatric patients with intracranial germ cell tumors is clinically important for radiation treatment and management. Partial/complete response is conventionally determined on postcontrast MR imaging sequences. The purpose of this study was to assess the diagnostic utility of a balanced steady-state free precession sequence as an adjunct to standard MR imaging sequences for the detection of residual tumor in pediatric patients on postchemoreduction pre-radiation planning MR imaging. MATERIALS AND METHODS: This was a retrospective study of pediatric patients with intracranial germ cell tumors undergoing postchemotherapy, preradiotherapy MR imaging. Patients underwent 1.5T or 3T MR imaging with pre- and postcontrast T1WIs, T2WIs, and a balanced steady-state free precession sequence. Two neuroradiologists independently reviewed standard MR imaging sequences without the balanced steady-state free precession sequence, then with the balanced steady-state free precession sequence 1 week later. Assessment for partial/complete response was determined using Response Assessment in Neuro-Oncology criteria. A 5-point Likert scale scored the diagnostic confidence of the neuroradiologist rating each study without/with the balanced steady-state free precession sequence. Rates of residual disease concordance and diagnostic confidence levels without/with the balanced steady-state free precession sequence were calculated. RESULTS: Thirty-nine patients were included with 31 males and 8 females (mean age, 14.15 ± 4.26 years). Thirty-one patients had single-site disease; 8 patients had multisynchronous disease (47 sites in total). Compared to review of the standard MR sequences alone, the addition of the balanced steady state free precession sequence resulted in higher rates of tumor partial response categorization and greater diagnostic confidence levels (P < .001, P < .001). CONCLUSIONS: The balanced steady-state free precession sequence improves detection of residual chemotherapy-reduced intracranial germ cell tumors and increases diagnostic confidence of the neuroradiologist. The balanced steady-state free precession sequence may be an important adjunct to the standard MR imaging protocol for radiation planning.

A predominant Th1 polarization is present in synovial fluid of end-stage osteoarthritic knee joints: analysis of peripheral blood, synovial fluid and synovial membrane.

Thorough understanding of the complex pathophysiology of osteoarthritis (OA) is necessary in order to open new avenues for treatment. The aim of this study was to characterize the CD4+ T cell population and evaluate their activation and polarization status in OA joints. Fifty-five patients with end-stage knee OA (Kellgren-Lawrence grades III-IV) who underwent surgery for total knee arthroplasty (TKA) were enrolled into this study. Matched samples of synovial membrane (SM), synovial fluid (SF) and peripheral blood (PB) were analysed for CD3+ CD4+ CD8- T cell subsets [T helper type 1 (Th1), Th2, Th17, regulatory T cells] and activation status (CD25, CD69, CD45RO, CD45RA, CD62L) by flow cytometry. Subset-specific cytokines were analysed by cytometric bead array (CBA). SM and SF samples showed a distinct infiltration pattern of CD4+ T cells. In comparison to PB, a higher amount of joint-derived T cells was polarized into CD3+ CD4+ CD8- T cell subsets, with the most significant increase for proinflammatory Th1 cells in SF. CBA analysis revealed significantly increased immunomodulating cytokines [interferon (IFN)-γ, interleukin (IL)-2 and IL-10] in SF compared to PB. Whereas in PB only a small proportion of CD4+ T cells were activated, the majority of joint-derived CD4+ T cells can be characterized as activated effector memory cells (CD69+ CD45RO+ CD62L- ). End-stage OA knees are characterized by an increased CD4+ T cell polarization towards activated Th1 cells and cytokine secretion compared to PB. This local inflammation may contribute to disease aggravation and eventually perpetuate the disease process.

Nerve growth factor secretion in cultured enteric glia cells is modulated by proinflammatory cytokines.

The enteric nervous system is composed of neurones and glial cells. These enteric glia cells (EGC) appear to be essential for the maintenance of gut homeostasis and mucosal integrity. Neurotrophin nerve growth factor (NGF) also plays an important role for the gut integrity by regulating sensory and inflammatory processes in the intestines. Here, we demonstrate EGCs as one source of NGF and show increased levels of NGF mRNA/protein and tropomyosin receptor kinase A (TrkA) mRNA in cultured EGCs upon stimulation with proinflammatory cytokines and lipopolysaccharides. NGF is continuously secreted from cultured EGCs and proinflammatory cytokines and lipopolysaccharides stimulate the secretion of this neurotrophin in a time- and dose- dependent manner, whereas interleukin-4 had no effect on NGF expression. Furthermore, NGF secretion was sustained for more than 12 h after withdrawal of the proinflammatory cytokines, suggesting the involvement of transcriptional and/or translational processes. Thus, the release of proinflammatory cytokines can increase NGF secretion by EGCs and leads to a higher expression of TrkA in EGCs. NGF, in turn, can increase visceral sensitivity and, on the other hand, appears to improve gut inflammation. Therefore, NGF secreting EGCs may play a key role in modulating visceral sensitivity and might be involved in inflammatory processes of the gut.

Recruitment of TRADD, FADD, and caspase 8 to double-stranded RNA-triggered death inducing signaling complexes (dsRNA-DISCs).

Rapid elimination of virus-infected cells by apoptosis is an efficient anti-viral strategy. Double-stranded RNA (dsRNA), a viral product, is potently and rapidly apoptogenic in susceptible cells. Caspase 8 plays an important role in the dsRNA-induced apoptosis; however, the mechanisms of caspase 8 activation in response to dsRNA are unknown. We demonstrate here that, in HeLa cells, the dsRNA-triggered activation of caspase 8 is independent of ongoing proteins synthesis (and is, therefore, independent of changes in pro- and anti-apoptotic gene expression) and involves the formation of multiprotein dsRNA-triggered death inducing signaling complexes (dsRNA-DISCs). DsRNA-DISCs contain FADD, TRADD, and caspase 8; however, several experimental approaches suggest that death ligands and death receptors (such as Fas/Apo1 and DR4/Apo2) are not involved in the formation of dsRNA-DISCs.

A modified presynchronization protocol improves fertility to timed artificial insemination in lactating dairy cows.

To compare 2 hormonal protocols for submission of lactating dairy cows for timed artificial insemination (TAI), nonpregnant lactating Holstein cows (n = 269) >60 d in milk were randomly assigned to each of 2 treatments to receive TAI (TAI = d 0). Cows assigned to the first treatment (Ovsynch, n = 134) received 50 microg of GnRH (d -10), 25 mg of PGF2alpha (d -3), and 50 microg of GnRH (d -1) beginning at a random stage of the estrous cycle. Cows assigned to the second treatment (Presynch, n = 135) received Ovsynch but with the addition of 2 PGF2alpha (25 mg) injections administered 14 d apart beginning 28 d (d -38 and -24) before initiation of Ovsynch. All cows received TAI 16 to 18 h after the second GnRH injection. Ovulatory response after each GnRH injection for a subset of cows (n = 109) and pregnancy status 42 d after TAI for all cows were assessed using transrectal ultrasonography. Based on serum progesterone (P4) profiles determined for a subset of cows (n = 109), P4 concentrations decreased for Presynch cows after the first 2 PGF2alpha injections, and Presynch cows had greater P4 concentrations at the PGF2alpha injection on d -3 compared with Ovsynch cows. Although the proportion of cows ovulating after the first and second GnRH injections did not differ statistically between treatments (41.1 and 69.6% vs. 35.9 and 81.1% for Ovsynch vs. Presynch, respectively), pregnancy rate per artificial insemination (PR/AI) at 42 d post TAI was greater for Presynch than for Ovsynch cows (49.6 vs. 37.3%). Parity, DIM, and body condition score (BCS) at TAI did not affect PR/AI to TAI. These data support use of this presynchronization protocol to increase PR/ AI of lactating dairy cows receiving TAI compared with Ovsynch.

Proinflammatory cytokines increase glial fibrillary acidic protein expression in enteric glia.

BACKGROUND: Enteric glia protect the integrity of the gut, as loss of enteric glial fibrillary acidic protein (GFAP) positive (+) glia leads to a haemorrhagic jejunoileitis. Crohn's disease (CD) and necrotising enterocolitis (NEC) show pathological changes in enteric glia. Therefore, factors controlling GFAP+ enteric glia are of great interest. The aim of the present study was to characterise enteric glia and determine the effect of interleukin 1beta (IL-1beta), interleukin 4 (IL-4), tumour necrosis factor alpha (TNF-alpha), and lipopolysaccharides (LPS) on cultured enteric glia. METHODS: Dissected rat colon and cultured enteric glia cells were double labelled with anti-GFAP and anti-S-100 antibodies. For regulatory studies, enteric glia cells were treated with cytokines and LPS. Proliferation was assayed using bromodeoxyuridine (BrdU) and mitosis of enteric glia was blocked by demecolcine. RESULTS: We were able to distinguish GFAP negative (-) from GFAP+ glia subtypes in situ and in primary cultures. Incubation of cells with IL-1beta, TNF-alpha, and LPS led to a significant increase in GFAP+ enteric glia while IL-4 had no effect on GFAP expression. After incubation with IL-1beta, total intracellular GFAP of enteric glia cells was increased. Upregulation of GFAP+ enteric glia could also be observed after stimulation with IL-1beta on blocking mitosis. BrdU uptake in stimulated enteric glia showed no increased proliferation rate. CONCLUSIONS: Two different types of enteric glia based on GFAP expression exist in the gut. Proinflammatory cytokines and LPS cause a dramatic increase in GFAP+ enteric glia. This suggests that cytokines play an important role in controlling GFAP+ enteric glia which might in turn be involved in modulating the integrity of the bowel during inflammation.

Glutamate 47 in 1-aminocyclopropane-1-carboxylate synthase is a major specificity determinant.

Glutamate 47 is conserved in 1-aminocyclopropane-1-carboxylate (ACC) synthases and is positioned near the sulfonium pole of (S,S)-S-adenosyl-L-methionine (SAM) in the modeled pyridoxal phosphate quinonoid complex with SAM. E47Q and E47D constructs of ACC synthase were made to investigate a putative ionic interaction between Glu47 and SAM. The k(cat)/K(m) values for the conversion of (S,S)-SAM to ACC and methylthioadenosine (MTA) are depressed 630- and 25-fold for the E47Q and E47D enzymes, respectively. The decreases in the specificity constants are due to reductions in k(cat) for both mutant enzymes, and a 5-fold increase in K(m) for the E47Q enzyme. Importantly, much smaller effects were observed for the kinetic parameters of reactions with the alternate substrates L-vinylglycine (L-VG) (deamination to form alpha-ketobutyrate and ammonia) and L-alanine (transamination to form pyruvate), which have uncharged side chains. L-VG is both a substrate and a mechanism-based inactivator of the enzyme [Feng, L., and Kirsch, J. F. (2000) Biochemistry 39, 2436-2444], but the partition ratio, k(cat)/k(inact), is unaffected by the Glu47 mutations. ACC synthase primarily catalyzes the beta,gamma-elimination of MTA from the (R,S) diastereomer of SAM to produce L-VG [Satoh, S., and Yang, S. F. (1989) Arch.Biochem. Biophys. 271, 107-112], but catalyzes the formation of ACC to a lesser extent via alpha,gamma-elimination of MTA. The partition ratios for (alpha,gamma/beta,gamma)-elimination on (R,S)-SAM are 0.4, < or =0.014, and < or =0.08 for the wild-type, E47Q, and E47D enzymes, respectively. The results of these experiments strongly support a role for Glu47 as an anchor for the sulfonium pole of (S,S)-SAM, and consequently a role as an active site determinant of reaction specificity.

The human cDNA for a homologue of the plant enzyme 1-aminocyclopropane-1-carboxylate synthase encodes a protein lacking that activity.

The sequences of genes encoding homologues of 1-aminocyclopropane-1-carboxylate (ACC) synthase, the first enzyme in the two-step biosynthetic pathway of the important plant hormone ethylene, have recently been found in Fugu rubripes and Homo sapiens (Peixoto et al., Gene 246 (2000) 275). ACC synthase (ACS) catalyzes the formation of ACC from S-adenosyl-L-methionine. ACC is oxidized to ethylene in the second and final step of ethylene biosynthesis. Profound physiological questions would be raised if it could be demonstrated that ACC is formed in animals, because there is no known function for ethylene in these organisms. We describe the cloning of the putative human ACS (PHACS) cDNA that encodes a 501 amino acid protein that exhibits 58% sequence identity to the putative Fugu ACS and approximately 30% sequence identity to plant ACSs. Purified recombinant PHACS, expressed in Pichia pastoris, contains bound pyridoxal-5'-phosphate (PLP), but does not catalyze the synthesis of ACC. PHACS does, however, catalyze the deamination of L-vinylglycine, a known side-reaction of apple ACS. Bioinformatic analysis indicates that PHACS is a member of the alpha-family of PLP-dependent enzymes. Molecular modeling data illustrate that the conservation of residues between PHACS and the plant ACSs is dispersed throughout its structure and that two active site residues that are important for ACS activity in plants are not conserved in PHACS.

[The Longo and Milligan-Morgan hemorrhoidectomy. A prospective comparative study of 300 patients]. / Hämorrhoidektomien nach Longo und Milligan-Morgan. Prospektive Vergleichsstudie mit 300 Patienten.

INTRODUCTION: Stapled haemorrhoidectomy according to Longo for treating reducible anal and mucosal prolapse appears to be very simple technically. In order to investigate the feasibility and to delineate the value of this procedure it was compared to standard open haemorrhoidectomy according to Milligan-Morgan in a prospective study. METHOD: In 1998 and 1999, 300 patients with third-degree haemorrhoids were operated on either with a Milligan-Morgan or a Longo technique. Intraoperative and postoperative performance--complications, length of surgery, consumption of analgetics, hospital stay, return to work--were evaluated. Follow-up was 1, 3 and 6 months. RESULTS: Length of surgery for both types of operation was the same. In comparison with the conventional open excision, patients with a stapled haemorrhoidectomy required considerably less analgetics. Hospital stay was shorter and return to work quicker and there were fewer morphological residues, e.g. skin tags. However, the costs of the procedure were considerably higher because of the disposable instrument. CONCLUSION: This new surgical procedure of supra-anodermal resection according to Longo offers advantages in the repair of prolapsing haemorrhoidal disease. Despite its primary simplicity, in our opinion the surgeon has to have colorectal, especially proctologic, experience. Although costs are significantly higher, this procedure might replace conventional techniques in many cases with prolapsing haemorrhoids.

Aminotransferase activity and bioinformatic analysis of 1-aminocyclopropane-1-carboxylate synthase.

The mechanistic fate of pyridoxal phosphate (PLP)-dependent enzymes diverges after the quinonoid intermediate. 1-Aminocyclopropane-1-carboxylate (ACC) synthase, a member of the alpha family of PLP-dependent enzymes, is optimized to direct electrons from the quinonoid intermediate to the gamma-carbon of its substrate, S-adenosyl-L-methionine (SAM), to yield ACC and 5'-methylthioadenosine. The data presented show that this quinonoid may also accept a proton at C(4)' of the cofactor to yield alpha-keto acids and the pyridoxamine phosphate (PMP) form of the enzyme when other amino acids are presented as alternative substrates. Addition of excess pyruvate converts the PMP form of the enzyme back to the PLP form. C(alpha)-deprotonation from L-Ala is shown by NMR-monitored solvent exchange to be reversible with a rate that is less than 25-fold slower than that of deprotonation of SAM. The rate-determining step for transamination follows the formation of the quinonoid intermediate. The rate-determining step for alpha, gamma-elimination from enzyme-bound SAM is likewise shown to occur after C(alpha)-deprotonation, and the quinonoid intermediate accumulates during this reaction. BLAST searches, sequence alignments, and structural comparisons indicate that ACC synthases are evolutionarily related to the aminotransferases. In agreement with previously published reports, an absence of homology was found between the alpha and beta families of the PLP-dependent enzyme superfamily.

Pericardial effusion associated with metastatic disease from an unknown primary tumor in a dog.

A 6.5-year-old, spayed female Siberian husky presented with signs of cardiac tamponade and weakness. Pleural, pericardial, and abdominal effusion were identified with radiographs and ultrasound. Pericardiocentesis relieved signs of tamponade, and the dog was clinically improved. Pericardial effusion recurred, and pericardiectomy was performed. Histopathological examination of excised tissues failed to reveal evidence of infectious or neoplastic disease. After pericardiectomy, clinically apparent thoracic effusion persisted. The dog was euthanized, and postmortem histopathological examination revealed emboli of metastatic carcinoma cells in the epicardium. The location of intrathoracic disease in this dog made antemortem diagnosis difficult, if not impossible.

L-Vinylglycine is an alternative substrate as well as a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate synthase.

L-Vinylglycine (L-VG) has been shown to be a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate (ACC) synthase [Satoh, S., and Yang, S. F. (1989) Plant Physiol. 91, 1036-1039] as well as of other pyridoxal phosphate-dependent enzymes. This report demonstrates that L-VG is primarily an alternative substrate for the enzyme. The L-VG deaminase activity of ACC synthase yields the products alpha-ketobutyrate and ammonia with a k(cat) value of 1.8 s(-1) and a K(m) value of 1.4 mM. The k(cat)/K(m) of 1300 M(-1) s(-1) is 0.17% that of the diffusion-controlled reaction with the preferred substrate, S-adenosyl-L-methionine. The enzyme-L-VG complex partitions to products 500 times for every inactivation event. The catalytic mechanism proceeds through a spectrophotometrically detected quinonoid with lambda(max) of 530 nm, which must rearrange to a 2-aminocrotonate aldimine to yield final products. Alternative mechanisms for the inactivation reaction are presented, and the observed kinetics for the full reaction course are satisfactorily modeled by kinetic simulation. The inactive enzyme is an aldimine with lambda(max) of 432 nm. It is resistant to NaBH(3)CN but is reduced by NaBH(4). ACC synthase is now expressed in Pichia pastoris with an improved yield of 10 mg/L.

Structure of 1-aminocyclopropane-1-carboxylate synthase, a key enzyme in the biosynthesis of the plant hormone ethylene.

The 2.4 A crystal structure of the vitamin B6-dependent enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase is described. This enzyme catalyses the committed step in the biosynthesis of ethylene, a plant hormone that is responsible for the initiation of fruit ripening and for regulating many other developmental processes. ACC synthase has 15 % sequence identity with the well-studied aspartate aminotransferase, and a completely different catalytic activity yet the overall folds and the active sites are very similar. The new structure together with available biochemical data enables a comparative mechanistic analysis that largely explains the catalytic roles of the conserved and non-conserved active site residues. An external aldimine reaction intermediate (external aldimine with ACC, i.e. with the product) has been modeled. The new structure provides a basis for the rational design of inhibitors with broad agricultural applications.

The neurotransmitter receptor-anchoring protein gephyrin reconstitutes molybdenum cofactor biosynthesis in bacteria, plants, and mammalian cells.

The molybdenum cofactor (Moco), a highly conserved pterin compound complexing molybdenum, is required for the enzymatic activities of all molybdenum enzymes except nitrogenase. Moco is synthesized by a unique and evolutionarily old pathway that requires the activities of at least six gene products. Some of the proteins involved in bacterial, plant, and invertebrate Moco biosynthesis show striking homologies to the primary structure of gephyrin, a polypeptide required for the clustering of inhibitory glycine receptors in postsynaptic membranes in the rat central nervous system. Here, we show that gephyrin binds with high affinity to molybdopterin, the metabolic precursor of Moco. Furthermore, gephyrin expression can reconstitute Moco biosynthesis in Moco-deficient bacteria, a molybdenum-dependent mouse cell line, and a Moco-deficient plant mutant. Conversely, inhibition of gephyrin expression by antisense RNA expression in cultured murine cells reduces their Moco content significantly. These data indicate that in addition to clustering glycine receptors, gephyrin also is involved in Moco biosynthesis and illustrate the remarkable conservation of its function in Moco biosynthesis throughout phylogeny.

The effect of the prone position on pulmonary mechanics is frame-dependent.

UNLABELLED: By compressing the abdomen and restricting chest wall movement, the prone position compromises pulmonary compliance. For spine surgery, placing the anesthetized patient into the prone position increases the risk of improper ventilation. In this study, we tested the hypothesis that the compromise in pulmonary compliance is related to the patient's body habitus and the surgical frame used to support the patient while in the prone position. Seventy-seven adult patients were divided into three groups according to body mass index: normal (n = 36) < or = 27 kg/m2, heavy (n = 21) 28-31 kg/m2, and obese (n = 20) > or = 32 kg/m2. Patients were placed in the prone position supported by chest rolls, a Wilson frame, or the Jackson spinal surgery table (Jackson table) according to the surgeon's preferences. Peak airway pressure (at the proximal endotracheal tube), pleural pressure (esophageal balloon), and mean arterial pressure were recorded in the supine position and prone position within 15 min of the turn. Dynamic mean (+/- SD) pulmonary compliance (mL/cm H2O) decreased when turning from the supine to the prone position in all three body mass groups when using chest rolls (normal 37+/-5 to 29+/-6; heavy 43+/-2 to 34+/-4; obese 42+/-8 to 32+/-6) or the Wilson frame (normal 39+/-6 to 32+/-7; heavy 43+/-16 to 34+/-10; obese 36+/-11 to 28+/-9). The dynamic pulmonary compliance was not altered in patients positioned on the Jackson table. Regardless of body habitus, using the Jackson table for prone positioning was not associated with a significant alteration in pulmonary or hemodynamic variables. We conclude that moving patients from the supine to the prone position during anesthesia results in a decrease in pulmonary compliance that is frame-dependent but that is not affected by body habitus. IMPLICATIONS: We hypothesized that compromise in pulmonary compliance in the prone position is related to the patient's body mass index and the surgical frame used. In this study, we demonstrated that prone positioning during anesthesia results in a decrease in pulmonary compliance that is frame-dependent but that is not affected by body mass index.

Elevated density and plasticity of nerve fibres in anal fissures.

Neural proliferative processes are regarded as a contributing factor in chronic inflammatory diseases and chronic pain. To elucidate whether neural proliferations occur in tissues surrounding chronic anal fissures and in the normal anal canal, the nerve fibre density was examined with the pan-neural marker protein gene product 9.5 (PGP) and the neural proliferative marker growth-associated protein 43 (GAP) by immunohistochemistry. GAP-immunoreactive nerve fibres in the uninflamed anal canal were distributed region specifically. The proportion of GAP-immunoreactive nerves in relation to the PGP-immunoreactive innervation exhibited regional differences. In tissue sections of chronic anal fissures, a marked increase in the density of PGP- and GAP-immunoreactive nerve fibres was noted, and PGP- and GAP-immunopositive nerve fibres displayed a neuroma-like appearance. Image analysis revealed that PGP- and GAP-immunoreactive innervation represented an area fraction of 0.5% (0.49 +/- 0.052; mean and SEM) and 0.1% (0.11 +/- 0.013) in the normal anal canal, respectively. In tissue sections of chronic anal fissures, PGP- and GAP-immunostained nerve fibres represented area fractions of 1.3% (1.32 +/- 0.12) and 0.6% (0.56 +/- 0.15), respectively. The increases in PGP- and GAP-immunopositive area fractions were highly significant (P > 0.01). The mean ratio of GAP to PGP immunoreactivities was not significantly increased in chronic anal fissures. The increase in pan-neural innervation and neuronal GAP immunoreactivity in tissues of anal fissures may imply that neuronal proliferation is involved in the pathogenesis of anal fissures. Neuronal proliferations may also be responsible for pruritus and severe pain in chronic anal fissures.