Nonselective matrix metalloproteinase but not tumor necrosis factor-α inhibition effectively preserves the early critical colon anastomotic integrity.

BACKGROUND: Increased matrix metalloproteinase (MMP) activity has been implicated in the pathogenesis of colorectal anastomotic leakage. Tumor necrosis factor-α (TNF-α) induces MMPs and may influence anastomosis repair. METHODS: We assessed the efficacies of the nonselective hydroxamate MMP inhibitor GM6001, the selective hydroxamate MMP inhibitor AG3340 and a TNF-α antagonist with respect to anastomotic breaking strength of left-sided colon anastomoses in male Sprague-Dawley rats. RESULTS: Systemic GM6001 treatment effectively blocked MMP activity and maintained the initial breaking strength day 0 of the anastomoses when administered subcutaneously as daily depositions (100 mg/kg) or continuously (10 mg/kg/day). In contrast, the anastomotic biomechanic strength was lowered by 55% (p < 0.001) in vehicle-treated rats on postoperative day 3. GM6001 treatment increased breaking strength by 88% (p < 0.0005) compared with vehicle-treated rats day 3 and reduced (p = 0.003) the occurrence of spontaneous anastomotic dehiscence. Histologically, the anastomotic wound was narrower (p < 0.05) in the longitudinal direction in GM6001-treated animals whereas GM6001 had no significant effect on inflammatory cell infiltration or epithelialization. AG3340 (10 mg/kg) increased (p < 0.012) breaking strength by 47% compared with vehicle on day 3 but did not significantly prevent the reduction of the initial breaking strength on day 0. Although the increased TNF-α levels in the wound were attenuated, the anastomotic breaking strength was not improved (p = 0.62) by the TNF-α (10 mg/kg) inhibitor given systemically. CONCLUSIONS: Pharmacological nonselective MMP inhibition ought to be explored as a prophylactic regimen to reduce anastomotic complications following colorectal resection. The involvement of TNF-α was insignificant in anastomotic wound healing in an experimental model.

Dietary alpha-ketoglutarate reduces gastrectomy-evoked loss of calvaria and trabecular bone in female rats.

OBJECTIVE: Surgical removal of the stomach (gastrectomy, Gx) leads to osteopenia in animals and in humans. In the rat, Gx adversely affects calvaria and trabecular bone. alpha-Ketoglutarate (AKG) is a precursor of hydroxyproline--the most abundant amino acid in bone collagen. The purpose of this study was to investigate the effects of dietary AKG on Gx-induced osteopenia. MATERIAL AND METHODS: Twenty female Sprague-Dawley rats were subjected to Gx and divided between two groups: Gx+AKG in the drinking water and Gx+Vehicle (i.e. drinking water without AKG). Another 20 rats were sham-operated and divided between two groups: Sham+AKG and Sham+Vehicle. The daily dose of AKG was 0.43 g per 100 g rat. All the rats were killed 8 weeks later and the calvariae, femora and tibiae were collected. The integrity of the calvariae was analysed planimetrically, following transillumination and photography. The bone mineral content (BMC) and bone mineral density (BMD) were measured in the right femorae and tibiae (bone densitometry), leaving the left femorae and tibiae to be analysed histomorphometrically (measurement of trabecular bone volume and trabecular fractal dimension). RESULTS: Gx caused calvarial bone degradation, reduced trabecular bone (femur and tibia) and impaired trabecular architecture. In addition, Gx lowered the femoral/tibial BMC and BMD (mainly cortical bone). Dietary AKG counteracted the Gx-evoked impairment of calvaria and trabecular bone but failed to affect the BMC and the BMD in either sham- operated or Gx rats. CONCLUSIONS: Gx resulted in loss of calvarial, trabecular and cortical bone in the rat. AKG counteracted the effect of Gx on calvaria and trabecular bone but not on cortical bone.

Phenotypic screening of hepatocyte nuclear factor (HNF) 4-gamma receptor knockout mice.

Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-gamma. HNF4-gamma is expressed in the kidneys, gut, pancreas, and testis. The first level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-gamma(+/+)), the HNF4-gamma knockout (HNF4-gamma(-/-)) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-gamma(-/-) mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test.

G protein-coupled receptor 12 deficiency results in dyslipidemia and obesity in mice.

Obesity has been proposed to be a result of an imbalance in the physiological system that controls and maintains the body energy homeostasis. Several G-protein coupled receptors (GPCRs) are involved in the regulation of energy homeostasis. To investigate the importance of GPCR12, mice deficient of this receptor (GPCR12 KO) were studied regarding metabolism. Expression of GPCR12 was found primarily in the limbic and sensory systems, indicating its possible involvement in motivation, emotion together with various autonomic functions, and sensory information processing. GPCR12 KO mice were found to have higher body weight, body fat mass, lower respiratory exchange ratio (RER), hepatic steatosis, and were dyslipidemic. Neither food intake nor energy in faeces was affected in the GPCR12 KO mice. However, lower energy expenditure was found in the GPCR12 KO mice, which may explain the obesity. In conclusion, GPCR12 is considered important for the energy balance since GPCR12 KO mice develop obesity and have lower energy expenditure. This may be important for future drugs that target this receptor.

Action of matrix metalloproteinases at restricted sites in colon anastomosis repair: an immunohistochemical and biochemical study.

BACKGROUND: Dehiscence of colon anastomosis is a common, serious and potentially life-threatening complication after colorectal operation. In experimental models, impaired biomechanic strength of colon anastomoses is preventable by general inhibitors of matrix metalloproteinases (MMPs) and associated with collagen loss, which indicates a possible link between MMP-mediated collagen degradation and dehiscence. The precise localization of collagen degradation within the anastomotic area and the specific MMPs responsible are unknown. METHODS: We have analyzed distinct zones within anastomoses using a novel microdissection technique for collagen levels, collagenolytic activity exerted directly by endogenous proteinases, and MMP-8 and MMP-9 immunoreactivity and their collagenolytic activity. RESULTS: The most pronounced collagen loss was observed in the suture-holding zone, showing a 29% drop compared with adjacent micro-areas of 3-day-old anastomoses. Only this specific tissue compartment underwent a dramatic and significant increase in collagenolysis, amounting to a loss of 10% of existing collagen molecules in 24 hours, and was abolished by metalloproteinase inhibitors. The tissue surrounding suture channels was heavily infiltrated with CD68-positive histiocytes that expressed MMP-8 and to a lesser extent MMP-9. The collagenolytic effect of the interstitial collagenase MMP-8 was synergistically potentiated by the gelatinase MMP-9 when added to colon biopsies incubated in vitro. CONCLUSIONS: The unique finding of this study was that the specific tissue holding the sutures of a colon anastomosis lost the most collagen presumably through induction and activation of multiple MMPs that may explain the beneficial effects of treatment with non-selective MMP antagonists.

CaV2.3 calcium channels control second-phase insulin release.

Concerted activation of different voltage-gated Ca( (2+) ) channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type Ca(V)2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in Ca(V)2.3-knockout (Ca(V)2.3(-/-)) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca(2+) channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological Ca(V)2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca(2+) signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single Ca(V)2.3(-/-) beta cells. Ca(V)2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the Ca(V)2.3(-/-) mouse. We propose a specific role for Ca(V)2.3 Ca(2+) channels in second-phase insulin release, that of mediating the Ca(2+) entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation.

Pharmacological treatment of osteopenia induced by gastrectomy or ovariectomy in young female rats.

BACKGROUND: Both gastrectomy (GX) and ovariectomy (OVX) induce osteopenia in man and experimental animals. The present study addresses the question--can alendronate, estrogen or parathyroid hormone (PTH) be used to treat established GX- or OVX -evoked osteopenia? METHODS: Rats were GX-, OVX- or SHAM-operated 8 weeks before starting the treatment with drugs. Each group was then treated for 8 weeks with 50 microg/kg/day alendronate, 10 microg/kg/day estrogen or 75 microg/kg/day PTH(1-84); n = 8 rats/group. Peripheral Quantitative Computed Tomography (pQCT) was used to measure trabecular bone mineral density (BMD) and various cortical bone parameters. RESULTS: At killing, 16 weeks after surgery, GX and OVX rats had a greatly reduced trabecular BMD in the metaphysis of the distal femur (GX -44% and OVX -55%). Alendronate increased the trabecular BMD by 44% in GX rats and by 64% in OVX rats, while PTH increased it by 51% and 115%, respectively. However, estrogen increased the trabecular BMD in GX rats (35%), but not in OVX rats (15%, not significant). Cortical bone parameters were adversely (but moderately) affected by GX, but not by OVX or by treatment with the three drugs. INTERPRETATION: Alendronate, estrogen and PTH restored the trabecular bone loss in rats with an established GX-evoked osteopenia. In contrast, alendronate and PTH, but not estrogen, restored the trabecular bone loss after OVX. Hence, the mechanism underlying GX-evoked bone loss differs from that underlying OVX-evoked bone loss. The ability of alendronate, estrogen and PTH to reverse the GX-evoked osteopenia in the rat may be of clinical interest when dealing with bone loss in humans after GX.