Dick Delbro

Engineering microporosity in bacterial cellulose scaffolds.

The scaffold is an essential component in tissue engineering. A novel method to prepare three‐dimensional (3D) nanofibril network scaffolds with controlled microporosity has been developed. By placing paraffin wax and starch particles of various sizes in a growing culture of Acetobacter xylinum, bacterial cellulose scaffolds of different morphologies and interconnectivity were prepared. Paraffin particles were incorporated throughout the scaffold, while starch particles were found only in the outermost area of the resulting scaffold. The porogens were successfully removed after culture with bacteria and no residues were detected with electron spectroscopy for chemical analysis (ESCA) or Fourier transform infra‐red spectroscopy (FT–IR). Resulting scaffolds were seeded with smooth muscle cells (SMCs) and investigated using histology and organ bath techniques. SMC were selected as the cell type since the main purpose of the resulting scaffolds is for tissue engineered blood vessels. SMCs attached to and proliferated on and partly into the scaffolds. Copyright

Loperamide improves anal sphincter function and continence after restorative proctocolectomy.

The physiological and clinical effects of loperamide treatment versus placebo were investigated in a randomized, double-blind, crossover study in patients operated with restorative proctocolectomy. Sixteen patients operated with endoanal mucosectomy and a handsewn ileal pouch-anal anastomosis and 14 patients operated with abdominal proctocolectomy and stapling of the pouch to the top of the anal canal were studied. While loperamide treatment increased resting anal pressure in both groups of patients by approximately 20% (P<0.05), squeeze pressure was not affected. Loperamide did not affect pouch volume or contractility. Sensory thresholds and the recto/pouch-anal inhibitory reflex were not influenced by loperamide treatment. Clinical function was improved, with a reduced bowel frequency and an improved nighttime continence, with less soiling (P<0.05) as well as need to wear a protective pad.

Altered muscarinic receptor subtype expression and functional responses in cyclophosphamide induced cystitis in rats.

In the in vitro study, it was investigated whether the expression of muscarinic receptors and cholinergic responses were altered in the situation of experimental cystitis. Rats were treated with cyclophosphamide intraperitoneally and the bladders were excised 36-100 h later. Immunohistochemistry and immunoblotting showed all subtypes of the muscarinic receptor (M1-M5) to be present in the specimens from inflamed urinary bladders and controls. In the cyclophosphamide-treated rats, the expression of muscarinic M5 receptors was increased by more than 40 times (p<0.01; n=8) both in the smooth muscle and the urothelium. Both the maximal contractile response to carbachol and to a high potassium concentration was approximately halved in cyclophosphamide-treated tissues, whereas the reduction was substantially greater in response to low carbachol concentrations (<EC(50)). The administration of 4-DAMP inhibited the carbachol-induced contractile responses of inflamed strips less potently than of controls, whereas pirenzepine and methoctramine showed equipotency in the two groups. The nitric oxide synthase inhibitor l-NNA increased the contractile effect of carbachol in inflamed detrusor strips, while it had no effect in controls. Immunoblotting showed endothelial nitric oxide synthase (eNOS) to be up-regulated in cystitis, and immunohistochemistry revealed the change to occur in the urothelium and in the suburothelial layer. The alteration of cholinergic detrusor responses in cyclophosphamide-treated rats depends mainly on a general detriment of contractility but also on indirect effects possibly via nitric oxide synthesis. The most prominent histological alterations occurred in the urothelium in which muscarinic M5 receptors increased in particular. The study further underlines that the urothelium may play significant roles in the pathogenesis of urinary bladder disorders such as interstitial cystitis.

Evans Blue Permeation of Intestinal Mucosa in the Rat

The azo dye Evans blue (EB; molecular weight, 960.83) is widely used as an indicator of increased capillary permeability. In the present study, however, rat gut absorption of EB was investigated after dye instillation in either the small or large intestine. During a brief period of ether anaesthesia, EB was injected either into jejunal loops with a challenge period of 30 or 60 min or into a proximal and a distal colon loop with a challenge period of 30, 60, or 120 min. After the rats had been killed the intestinal specimens were washed with 6 mM acetylcysteine dissolved in phosphate-buffered saline, which efficiently cleared the tissues of mucus, and thus of EB trapped in mucus. Only EB absorbed by the gut wall remained to be estimated, and this absorption was found to be both dose- and time-dependent in the jejunum and the colon. After instillation in the colon, but not in jejunum, EB could be detected in the blood. EB absorption from the jejunum remained unaffected by the addition of either ouabain (1 mM) or lidocaine (0.38 mM). Either of these compounds inhibited EB uptake in the proximal part of the colon, while enhancing it in the distal part. Fluorescence microscopy showed penetration into the intestinal wall to be a prerequisite for EB to become fluorescent, and EB fluorescence increased with time. It is proposed that EB is transported over the mucosa by the paracellular route and that the amount of absorbed EB reflects epithelial permeability differently in different parts of the gastrointestinal tract.(ABSTRACT TRUNCATED AT 250 WORDS)

Preconditioning protects against ischemia/reperfusion injury of the liver

Ischemic preconditioning (IPC) of an organ may induce protection against the injury caused by longer duration of ischemia and subsequent reperfusion. In a standardized model of such injury in the rat liver, we used the following protocol to investigate whether adenosine played a role in IPC by preventing its enzymatic degradation by dipyridamole pretreatment according to the following protocol: group 1, non-ischemic control rats; group 2, ischemic control rats subjected to 60 minutes of ischemia by clamping of the common hepatic artery followed by 60 minutes of reperfusion; group 3, IPC with 10 minutes of ischemia followed by 15 minutes of reperfusion, prior to the ischemia/reperfusion period as in group 2; group 4, pharmacologic preconditioning with administration of dipyridamole prior to the ischemia/reperfusion period as in group 2. Peripheral liver blood flow was significantly reduced during clamping (groups 2 to 4). After unclamping, blood flow was still reduced in the ischemic rats (group 2) but had returned to preclamp values in the animals that had been subjected to ischemic (group 3) or pharmacologic (group 4) preconditioning. Liver cell injury was significantly increased in the ischemia group (group 2) only. In our experimental model of ischemia/reperfusion injury in the rat liver, we found an equally beneficial effect with ischemic and pharmacologic preconditioning. Adenosine appears to be a crucial factor in IPC.Ischemic preconditioning (IPC) of an organ may induce protection against the injury caused by longer duration of ischemia and subsequent reperfusion. In a standardized model of such injury in the rat liver, we used the following protocol to investigate whether adenosine played a role in IPC by preventing its enzymatic degradation by dipyridamole pretreatment according to the following protocol: group 1, non-ischemic control rats; group 2, ischemic control rats subjected to 60 minutes of ischemia by clamping of the common hepatic artery followed by 60 minutes of reperfusion; group 3, IPC with 10 minutes of ischemia followed by 15 minutes of reperfusion, prior to the ischemia/reperfusion period as in group 2; group 4, pharmacologic preconditioning with administration of dipyridamole prior to the ischemia/reperfusion period as in group 2. Peripheral liver blood flow was significantly reduced during clamping (groups 2 to 4). After unclamping, blood flow was still reduced in the ischemic rats (group 2) but had returned to preclamp values in the animals that had been subjected to ischemic (group 3) or pharmacologic (group 4) preconditioning. Liver cell injury was significantly increased in the ischemia group (group 2) only. In our experimental model of ischemia/reperfusion injury in the rat liver, we found an equally beneficial effect with ischemic and pharmacologic preconditioning. Adenosine appears to be a crucial factor in IPC.

The role of the Lps gene in experimental ulcerative colitis in mice

The effects of the Lps gene on the development of experimental ulcerative colitis were studied in two genetically different mouse strains: C57B1 and C3H. Acute colitis was induced by adding 3% dextran sulfate sodium (DSS) to the drinking water for a 7‐day (C57B1 and C3H) or a 10‐day (C57B1) experimental period. Although the DSS treatment initiated the same type of morphological changes in the colon in all groups of mice, an earlier onset and persistent intestinal bleeding occurred in the Lpsn mice (sensitive to lipopolysaccharide, LPS) in comparison with the Lpsd mice (hyporesponsive to LPS). Rectal bleeding appeared on day 7 in 90% of the Lpsn compared to 13% of the Lpsd mice (p<0.0001). In C57B1 mice, followed for three additional days, 50% of the Lpsn mice died and the surviving animals showed as well as rectal bleeding a large number of Gram‐negative bacteria in the liver and spleen. In contrast, the Lpsd mice of the C57B1 strain appeared unaffected by the treatment, although a transient rectal bleeding occurred in 90% on day 8. Also, significantly fewer Gram‐negative bacteria were found in the liver and spleen. Even though significantly increased serum endotoxin levels were seen in all DSS‐treated groups compared to controls on day 7, the serum levels of TNFoc were significantly increased only in the Lpsn mice. In DSS‐induced colitis the Lps“ genotype conferred on the mice an increased LPS susceptibility, resulting in an augmentation of the inflammatory response to Gram‐negative bacteria and their endotoxins. The results suggest that LPS‐induced host effector mechanisms significantly enhanced the intestinal bleeding, systemic inflammatory response, and mortality in mice with DSS‐induced colitis. In addition, the host defense against the invading and system‐ically spread bacteria most probably involved additional genes.

DMPP causes relaxation of rat distal colon by a purinergic and a nitrergic mechanism

The non-adrenergic relaxation of carbachol precontracted longitudinal muscle of the rat distal colon was investigated. Intrinsic nerves were activated by the nicotinic, ganglionic receptor agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP). DMPP at 1 and 4 microM caused a relaxation that was markedly antagonized by the nerve blocker tetrodotoxin (1 microM) or the nicotinic receptor antagonist, hexamethonium (1 mM). The response to DMPP was significantly antagonized by apamin (an inhibitor of ATP-sensitive K+-channels), by reactive blue 2 (a blocker of P2y purinoceptors) and by an inhibitor of nitric oxide (NO)-synthase (N(G)-nitro-L-arginine, L-NNA). The combined treatment with reactive blue 2 and L-NNA reduced the relaxatory response to 1 microM DMPP by 77 +/- 8% and to 4 microM DMPP by 58 +/- 4% of control, but left a residual component. Our results indicate that ATP and NO, together with at least one additional (hitherto unidentified) substance may be inhibitory neurotransmitters in rat distal colon.

Tonic inhibition of small intestinal motility by nitric oxide.

The effects of blocking nitric oxide synthase with the arginine analog N omega-nitro-L-arginine (L-NNA) were investigated in anaesthetized cats, vagotomized and pretreated with guanethidine and atropine. Spontaneous NANC jejunal motility (recorded as the volume changes of an intraluminal balloon) was markedly increased in a dose-dependent and stereospecific manner. The effect of L-NNA was partly reversed by L-arginine, the substrate for nitric oxide (NO) synthesis. Thus, this study presents evidence for a tonic inhibitory influence, via the release of NO, on small intestinal motility in vivo. Furthermore, relaxations upon the L-NNA-induced hypermotility could be elicited by vagal nerve stimulation, which may suggest the existence of another NANC inhibitory transmitter. Hexamethonium abolished such relaxations but did not affect the tone or phasic activity after L-NNA.

Role of nitric oxide in motility and secretion of the feline hepatobiliary tract.

BACKGROUND Nitric oxide (NO) mediates inhibition of gastrointestinal smooth-muscle cells via nonadrenergic, non-cholinergic (NANC) nervous pathways. The effect of NO on the absorption and secretion by the mucosa of the gastrointestinal and hepatobiliary tracts is less well known. The aim of this study was to evaluate the effects of a pharmacologic blockade of NO synthase on sphincter of Oddi activity, gallbladder function, and bile secretion and to demonstrate the presence of NO synthase-positive neurons in this region. METHODS Experiments were conducted on anesthetized cats after blockage of noradrenergic and cholinergic neurotransmission. Flow resistance in the sphincter of Oddi, gallbladder fluid absorption and motility, bile outflow from the liver, and bile salt secretion were registered. RESULTS Flow resistance exerted by the sphincter of Oddi increased dose-dependently in response to the NO synthase blocker NG-nitro-L-arginine. The increase in flow resistance was reversed stereospecifically by L-arginine, the substrate for NO synthesis. No significant effects on bile secretion, gallbladder fluid transport, or gallbladder motility were observed. NO synthase-positive neurons were identified close to the sphincter of Oddi and in the gallbladder mucosa. CONCLUSIONS This tonically active inhibitory NANC innervation of the sphincter of Oddi may be important in the physiologic regulation of the bile duct pressure.

Is acetylcholine an autocrine/paracrine growth factor via the nicotinic α7-receptor subtype in the human colon cancer cell line HT-29?

We used immunochemistry to demonstrate expression of acetylcholines nicotinic alpha7-receptor subtype in human colon cancer cell line HT-29. Moreover, RT-PCR and immunochemistry showed that choline acetyltransferase and acetylcholine esterase, the enzymes responsible for acetylcholine synthesis and degradation, respectively, localise in HT-29 cells. Bromoacetylcholine bromide, an inhibitor of choline acetyltransferase, significantly attenuated basal cell growth. Our findings suggest that acetylcholine might serve as an autocrine/paracrine-or speculatively, even intracrine-signalling molecule in cell line HT-29, thus contributing to carcinogenesis/cancer progression.