Poly(alpha-hydroxyacids) for application in the spinal cord: resorbability and biocompatibility with adult rat Schwann cells and spinal cord

Future surgical strategies to restore neurological function in the damaged human spinal cord may involve replacement of nerve tissue with cultured Schwann cells using biodegradable guiding implants. We have studied the in vitro and in vivo degradability of various aliphatic polyesters as well as their effects on rat Schwann cells in vitro and on spinal cord tissue in vivo. In vitro, cylinders made of poly(D,L-lactic-co-glycolic acid) 50:50 (PLA25GA50) started to degrade at 7 days, compared with 28 days for cylinders made of poly(D,L-lactic acid) (PLA50). This faster degradation of PLA25GA50 was reflected by a much higher absorption of water. In vivo, after implantation of PLA25GA50 or PLA50 cylinders between the stumps of a completely transected adult rat spinal cord, the decrease in molecular weight of both polymers was similar to that found in vitro. In vitro degradation of poly(L-lactic acid) (PLA100) mixed with increasing amounts of PLA100 oligomers also was determined. The degradation rate of PLA100 mixed with 30% oligomers was found to be similar to that of PLA50. In vitro, PLA25GA50 and the breakdown products had no adverse effect on the morphology, survival, and proliferation of cultured rat Schwann cells. In vivo, PLA25GA50 cylinders were integrated into the spinal tissue 2 weeks after implantation, unlike PLA50 cylinders. At all time points after surgery, the glial and inflammatory response near the lesion site was largely similar in both experimental and control animals. At time points later than 1 week, neurofilament-positive fibers were found within PLA25GA50 cylinders or the remains thereof. Growth-associated protein 43, which is indicative of regenerating axons, was observed in fibers in the vicinity of the injury site and in the remains of PLA25GA50 cylinders. The results suggest that poly(alpha-hydroxyacids) are likely candidates for application in spinal cord regeneration paradigms involving Schwann cells.     

In vivo model to study the biocompatibility of peritoneal dialysis solutions

This study was designed to analyze the complex morphologic and functional effects of dialysis solutions on peritoneum in a rat model on chronic peritoneal dialysis. Peritoneal catheters were inserted into 10 male, Wistar rats and the animals were dialyzed twice daily for 4 weeks with 4.25% Dianeal. During the study we observed two opposite effects: healing of the peritoneum after catheter implantation--decreased cell count in dialysate, decreased permeability of the peritoneum to glucose and total protein, increased volume of drained dialysate; and damage to the membrane due to its exposure to peritoneal dialysis solution--increased hyaluronic acid levels in dialysate, a tendency of the peritoneum to thicken when compared to non-dialyzed animals. Our rat model of CAPD may be used for quantitative and qualitative assessment of the effects of peritoneal dialysis solution on the peritoneum during chronic dialysis.     

Poly (lactic-glycolic) acid copolymer encapsulation of recombinant adenovirus reduces immunogenicity in vivo

Adenovirus-mediated gene transfer has application to the treatment of diseases of the central nervous system. We demonstrate that a limitation to its use in vivo is an inability to redose to the brain. We show that one factor inhibiting re-dosing is the development of neutralizing anti-adenoviral antibodies. Encapsulation of recombinant adenovirus vectors in poly(lactic/glycolic acid) (PLGA) copolymer enables infection in vitro, in the presence of neutralizing antibodies and results in the release of viable virus for over 100 h. Importantly, encapsulated adenovirus also shows diminished immunogenicity in vivo. Mice immunized with encapsulated recombinant adenoviral vectors show a greater than 45-fold reduction in anti-adenovirus titers relative to non-encapsulated vectors. An extended release formulation of adenovirus that reduces viral immunogenicity and sequesters the viral particle form antibody exposure may improve in vivo efficacy.     

Evaluation of the hairless rat as a model for in vivo percutaneous absorption

Interleukin-10 (IL-10) is known to inhibit T cell-mediated responses. IL-10 has also been shown to play an important pathogenetic role in allergic diseases. Glucocorticoid is known to inhibit the production and gene expression of many cytokines which induce inflammatory reactions. We examined the effect of dexamethasone on the gene expression and production of IL-10 by human peripheral blood mononuclear cells (PBMCs) and monocytes. PBMCs and monocytes from 5 healthy volunteers were incubated with or without dexamethasone for 1 h, then stimulated with 5 micrograms/ml lipopolysaccharide (LPS). Gene expression and production of IL-10 by human PBMCs were detected without stimulation and increased by LPS stimulation. Dexamethasone suppressed the gene expression and production of IL-10 by LPS-stimulated PBMCs in a dose-dependent manner by 41.6 and 61.1% at 10(-6) M, respectively. Also in monocytes, the gene expression and production of IL-10 were detected without stimulation, increased by LPS stimulation, and significantly suppressed by dexamethasone by 53.1 and 61.2% at 10(-6) M, respectively. This suppressive effect on IL-10 gene expression was not so potent compared with its effect on cytokines such as IL-5. The suppression of IL-10 production by glucocorticoid is suggested to be one of the important mechanisms by which glucocorticoids suppress allergic inflammation in the treatment of allergic diseases.     

Poly(alkyl cyanoacrylate) nanocapsules as a delivery system in the rat for octreotide, a long-acting somatostatin analogue

Poly(alkyl cyanoacrylate) nanocapsules have been used as biodegradable polymeric drug carriers for subcutaneous and peroral delivery of octreotide, a long-acting somatostatin analogue; their ability to reduce insulin secretion or prolactin secretion in response to oestrogens has been studied in adult male rats. The nanocapsules, prepared by interfacial emulsion polymerization of isobutyl cyanoacrylate, were 260 nm in diameter and incorporated 60% of octreotide. Administered subcutaneously, the octreotide-loaded (20 micrograms kg-1) nanocapsules suppressed the insulinaemia peak induced by intravenous glucose overload and depressed insulin secretion over 48 h, preventing the secretory rebound; however, glycaemia was unaffected. In parallel, the plasma octreotide concentration increased 2.7 times. Administered perorally to oestrogen-treated rats, octreotide-loaded nanocapsules (200 and 1000 micrograms kg-1) significantly improved the reduction of prolactin secretion (by 72 and 88%, respectively, compared with 32 and 54% with free octreotide) and slightly increased plasma octreotide level. Thus nanocapsules could be of interest as a biodegradable drug carrier for the administration of octreotide.     

In vivo metabolism of a novel cholecystokinin B (CCK-B) antagonist in rat and dog plasma and rat faeces

Human extracellular ribonucleases (RNase), together with other members of the mammalian RNase A superfamily, can be classified into four different RNase families on the basis of their structural, catalytic and/or biological properties. Their occurrence and main distinctive features have been described, and the information available on their catalytic properties has been analysed and discussed in comparison with those of other animal RNases. On the basis of some results obtained with various single- and double-stranded polyribonucleotides, it has been proposed that while pancreatic-type (pt) RNases could be defined as single-strand/pyrimidine 'preferring' ribonucleases, mammalian nonpancreatic-type (npt) RNases may be referred to as single-strand/pyrimidine 'specific' ribonucleases. In addition, some data concerning human nptRNases may support the suggestion [Cuchillo et al. (1993) FEBS Lett. 333: 207-210] that the enzyme 'ribonuclease' should be reclassified as 'transferase'.     

C-reactive protein (CRP) as a marker of of hemodialysis biocompatibility]

Haemodialysis leads to monocytes activation and secretion of cytokines, which stimulates hepatic production of CRP. To assess the biocompatibility of haemodialysis the CRP serum levels were measured. CRP serum levels during haemodialysis with the use of cuprophane membranes increased from 4743.3 +/- 3251.6 ng/ml to 5231.8 +/- 3458.4 ng/ml just after haemodialysis and 5865.4 +/- 3684.8 ng/ml 22 hours after haemodialysis (p < 0.001). During haemodialysis using polysulfone membranes CRP from the initial value of 4819.4 +/- 4328.2 ng/ml decreased to 3316.9 +/- 3882.7 ng/ml just after haemodialysis (p < 0.01) and increased to 5086.9 +/- 4193.0 ng/ml 22 hours after haemodialysis (p < 0.05). Re-counted CRP values, according to changes in total blood protein, increased significantly (p < 0.02) 22 hours after haemodialysis with the use of cuprophane membranes. During haemodialysis using polysulfone membranes above mentioned levels were significantly decreased just after haemodialysis (p < 0.001). The cuprophane membranes surface area and reutilization of dialyzers did not affect the changes of CRP serum levels. No correlation was observed between CRP level changes and dialysis neutropenia and complement activation. Our results indicate, that CRP serum level measurement may be feasible to assess the biocompatibility of dialysis membranes.     

Observations on seminal vesicle dynamics in an in vivo rat model

PURPOSE: To gain understanding of the seminal vesicle as a muscular organ, seminal vesicle compliance and contractile properties were quantified with an in vivo, microsurgical rat model. MATERIALS AND METHODS: Microsurgical dissection was performed on anesthetized rats to enable simultaneous organ filling and monitoring of intraluminal pressures. The reliability and reproducibility of post-ganglionic hypogastric nerve-induced ipsilateral (4 rats) and bilateral (5 rats) seminal vesicle contractile responses were assessed during repeated nerve stimulation. Seminal vesicle resting compliance was assessed during a constant saline infusion (10 rats). Functional performance curves were obtained at fixed fill-volumes by measuring organ contraction after nerve stimulation (4 rats). RESULTS: A reproducible seminal vesicle contractile response was obtained with a nerve stimulation interval > 15 minutes. Bilateral seminal vesicle responses were observed with unilateral nerve stimulation. The resting organ compliance curve with saline filling exhibited a characteristic, triphasic response. Functional performance studies revealed that contractile performance improves as the fill-volume increases until the distensibility limit of the organ is reached. CONCLUSIONS: A reliable, in vivo, rat model of seminal vesicle organ compliance and contractility is described. The seminal vesicle is a highly contractile, compliant smooth muscular organ with dynamic properties analogous to that of the urinary bladder. This experimental system may allow for the investigation of pharmacologic and other physiological influences on in vivo organ activity.     

Poly(2-aminoadenylic acid): interaction with poly(uridylic acid)

Poly(2-aminoadenylic acid) forms both double and triple helices with poly(uridylic acid) [poly(U)]. The 2-amino group forms a third hydrogen bond, elevating the 2 leads to 1 transition temperature by 33 degrees C. The third strand, however, has about the same stability as poly(A)-2poly(U), as measured by Tm 3 leads to 2. This selective stabilization of the two-stranded helix results in a much greater resolution of the differnt thermal transitions than that observed in analogous polynucleotide systems. In contrast to other A, U systems 3 leads to 1 and 2 leads to 3 transitions are not observed under any conditions, and the triple helix always undergoes a 3 leads to 2 transition even at very high ionic strength. A 1:1 mixture of poly(2NH2A) and poly(U) exhibits no transient formation of 1:2 complex, unlike similar mixtures of poly(A) with poly(U) and poly(T). This difference is evidently due to a more rapid displacement reaction: [poly(2NH2A) + poly(2NH2A)-2poly(U) leads to 2 poly(2NH2A)-poly(U)] With poly(2NH2A) than with poly(A). We describe a method for establishing the combining ratios of polynucleotide complexes which used a computer to calculate the angles of intersection of mixing curves as explicit and continuous functions of the wavelength. The wavelength dispersions of the angles of intersection determine optimum wavelengths for establishing stoichiometry and can also provide reliable negative evidence that presumably plausible complexes are not formed. Analogous computer procedures have been developed to determine wavelengths which are selective for the formation of both 1:1 and 1:2 complexes. Infrared spectra of the 1:1 and 1:2 complexes resemble those of other A, U homoribopolynucleotide helices in having two and three strong bands, respectively, in the region of carbonyl stretching vibrations. CD spectra of the two complexes are unusual in having negative first extrema of moderate intensity. We attribute these extrema to intrastrand interactions of strong, well-resolved transitions at 278 nm (B2u) of the 2-aminoadenine residues. The CD spectra are correlated with those of other polynucleotide helices.     

In vivo dexamethasone effects on neutrophil effector functions in a rat model of acute lung injury

1. In healthy male volunteers, the absorption, metabolite profiles and excretion of 14C-benidipine hydrochloride, a new Ca antagonist, were investigated after oral administration at a dose of 8 mg. 2. 14C-benidipine hydrochloride was rapidly absorbed, and the plasma concentration of radioactivity and unchanged drug reached a maximum of 71.2 ng eq./ml at 1.1 h and 2.56 ng/ml at 0.6 h respectively, and then declined bi-exponentially. The half-life in the elimination phase was 14.7 and 5.3 h respectively, AUC of unchanged drug was low, about 1% of that of radioactivity. 3. Five days after administration, 36.4% of the administered radioactivity was excreted in urine and 58.9% in faeces. 4. The metabolite profiles in plasma, urine and faeces were analysed by hplc. At 1 h after administration the predominant metabolites in plasma were M9 and M2, which accounted for 13.8 and 8.2% of the radioactivity respectively, whereas unchanged drug represented 1.2%. Predominant metabolites in urine 12 h after administration were M3 and M8, which accounted for 2.22 and 2.21% of the administered radioactivity respectively. Metabolites excreted in faeces 120 h after administration were very complex and poorly separated by hplc and could not be characterized: unchanged drug was not detected in the faeces.     

Differences in thermal salivation between the FOK rat (a model of genotypic heat adaptation) and three other rat strains

Rats secrete saliva in response to heat. In the present study, details of thermal salivation were investigated using the FOK rat in comparison with Sprague-Dawley (SD), Donryu, and ACI rats. The FOK rat is a strain inbred for genotypic heat adaptation and endures heat for long periods. Conscious rats of all four strains were exposed to 42.5 degrees C. The order of heat endurance times at this temperature was FOK > SD > Donryu = ACI. FOK rats spread their saliva over their entire ventral surface, their faces, and their outside legs. This saliva area was wider than those made by the other three strains. SD rats spread in an area wider than those of the Donryu and ACI rats. Saliva spreading in the FOK rats continued for 4.0-4.5 h, far longer than in the other strains. Under ketamine anesthesia and exposure to 40 degrees C, the FOK rats secreted saliva at 1390+/-235 microL/100 g of body weight during a 60-min observation period. This was the highest rate among the four rat strains (p < 0.0001). The body temperature increase rate in anesthetized FOK and SD rats was lower than in the other two strains, suggesting a minor contribution of unknown factors. Ligation of the submandibular gland ducts abolished the thermal salivation of the FOK rats, whereas ligation of the parotid duct had no effect. The submandibular, sublingual, and lachrymal glands in the FOK rats were 1.3-1.5, 1.25-1.4, and 1.3-1.5 times heavier, respectively, than those in the other three strains, whereas the parotid gland of the FOK rats was not enlarged. These findings indicate that the rats' saliva spreading and ET values are significantly correlated. A potentiated and long-lasting salivation from the submandibular gland was acquired during development of genotypic heat adaptation. This salivation is actuated in response to heat. The pronounced thermal salivation is probably attributable to adaptive changes in the superior salivatory nucleus-chorda tympani-submandibular gland pathway.     

A hydrogel based on a polyaspartamide: characterization and evaluation of in-vivo biocompatibility and drug release in the rat

This paper deals with the characterization of a new microparticulate hydrogel obtained by gamma irradiation of alpha, beta-poly[N-(2-hydroxyethyl)-DL-aspartamide] (PHEA). When enzymatic digestion of PHEA hydrogel was evaluated using various concentrations of pepsin and alpha-chymotrypsin no degradation occurred within 24 h. In-vivo studies showed that this new material is biocompatible after oral administration to rats. PHEA hydrogel was also studied as a system for delivery of diflunisal, an anti-inflammatory drug. In-vitro release studies in simulated gastrointestinal juice (pH 1 or 6.8) showed that most of the drug was released at pH 6.8. In-vivo studies indicated that diflunisal-loaded PHEA microparticles significantly improved the gastric tolerance and oral bioavailability of the drug in comparison with free diflunisal. These results suggest the potential application of PHEA hydrogel as a new delivery system for the oral administration of anti-inflammatory drugs.     

Poly(A) tail shortening by a mammalian poly(A)-specific 3'-exoribonuclease

3'-Exonucleolytic removal of the poly(A) tail is the first and often rate-limiting step in the decay of many eucaryotic mRNAs. In a cytoplasmic extract from HeLa cells, the poly(A) tail of mRNA was degraded from the 3'-end. In agreement with earlier in vivo observations, prominent decay intermediates differed in length by about 30 nucleotides. The Mg2+-dependent, poly(A)-specific 3'-exoribonuclease responsible for this poly(A) shortening activity was purified from calf thymus. A polypeptide of 74 kDa copurified with the activity. The deadenylating nuclease (DAN) required a free 3'-OH group, released solely 5'-AMP, degraded RNA in a distributive fashion, and preferred poly(A) as a substrate. At low salt concentration, the activity of purified DAN was strongly dependent on spermidine or other, yet unidentified factors. Under these reaction conditions, DAN was also stimulated by the cytoplasmic poly(A)-binding protein I (PAB I). At physiological salt concentration, the stimulatory effect of spermidine was weak and PAB I was inhibitory. At either salt concentration DAN and PAB I reconstituted poly(A) shortening with the same pattern of intermediates seen in cytoplasmic extract. The properties of DAN suggest that the enzyme might be involved in the deadenylation of mRNA in vivo.     

Poly(ethylene glycol) fluorescent linkers

The first examples of PEG linkers containing the highly fluorescent dansyl group have been synthesized. Quantum yields of these PEG fluorescent linkers (PFL) were determined and utilized in calculating the PEG number of various protein conjugates. The method was also shown to be applicable to lower molecular weight drugs as exemplified by taxol.     

The intact isolated (ex vivo) retina as a model system for the study of excitotoxicity

Excitotoxicity is defined as a mode of neural cell death triggered by overactivation of receptors for the amino acid transmitter glutamate. There is considerable evidence that excitotoxicity is responsible for cell death in several neuropathological states, including some retinal diseases. The isolated retina, particularly from chick embryos, has been used extensively as an experimental system to characterize this process. This paper summarizes the use of isolated retina as a model system for studies of excitotoxicity from a theoretical and methodological point of view, and reviews results obtained from studies utilizing this system.     

Poly(ADP-ribose) polymerase and aging

The activation of oncogenes and the inactivation of tumour suppressor genes play a critical role in laryngeal tumorigenesis. Recent investigations revealed that 8p, 9p and 17q arms of human chromosomes harbour tumour suppressor genes (TSGs) such as p16 and BRCA1 with an important role in the multistage carcinogenesis of the larynx. In order to investigate the implication of these novel TSGs in the development of laryngeal neoplasia we performed a loss of heterozygosity (LOH) analysis using a bank of 15 polymorphic microsatellite markers (4 at 8p21, 7 at 9p21 arm and 4 at 17q arm surrounding the BRCA1 region) in a series of 32 cytological specimens (19 squamous cell carcinoma, 13 benign lesions of the larynx). Both benign and malignant specimens exhibited genetic alterations with at least one microsatellite marker. Fifteen (47%) out of the 32 specimens exhibited LOH at 8p21, 25/32 (78%) showed LOH at 9p21 and 18/32 (56%) displayed LOH at 17q21. Genetic alterations were detected in both benign and malignant lesions for all the loci tested suggesting an important role of these regions in the development of laryngeal neoplasia. This is the first report of detection of microsatellite alterations not only in solid tumours of the larynx but in laryngeal cytological specimens, suggesting that microsatellite analysis may be a useful tool in the primary diagnosis of the disease.     

Divergence of beta-myosin heavy chain (betaMHC) expression in fetal rat cardiomyocytes in vitro and adult rat heart in vivo

The myosin heavy chain gene products are an important determinant of myocardial functional properties. Although a strong positive element (beta f1) within the betaMHC promoter region has previously been identified, to date no species comparisons in promoter strength have been made. To examine this question, we have used betaMHC deletion constructs, containing the rat or human beta f1 enhancer region, to determine expression both in vitro using rat fetal cardiomyocytes and in vivo by direct injection into adult rat heart. When reporter constructs were transfected into cultured fetal rat cardiomyocytes, the human beta reporter was expressed more than 3 fold above the equivalent rat construct. Exchange of the beta f1 enhancer indicated that the human sequence of the beta f1 enhancer was largely responsible. However, these findings were not replicated when the reporters were injected into the adult rat heart. In the adult myocardium the levels of reporter expression were similar for the betaMHC promoter reporters studied. These findings demonstrate a divergence between primary cardiomyocytes maintained in culture and the cardiomyocytes found within the intact adult heart.     

Allyl-containing Poly(aryl ether sulfone): Synthesis and Crosslinking

A novel type of crosslinkable poly(aryl ether sulfone)(PAES) bearing an allyl pendant(PES-OAllyl) was synthesized by a grafting reaction of hydrophenyl-containing PAES(PES-OH) and allyl bromide. PES-OH was pre- pared by a demethylation reaction of a methoxyphenylated PAES(PES-OCH<,3>) in the presence of pyridine/hydrochlo- ride. The PES-OCH<,3> was synthesized by an aromatic nucleophilic substitution of bis(4-chlorophenyl)sulfone and (p-methoxy)phenylhydroquinone. Both DSC and solubility investigation were used to study the crosslinking behavior of PES-OAllyl. After heat treatment, the glass transition temperature(T<,g>) value of PES-OAllyl sharply increased from 165 ℃ to 227 ℃. Meanwhile, PES-OAllyl changed from a soluble polymer to an insoluble thermoset. In addition, TGA(thermogravimetric analysis) result suggests that the thermal stability of the crosslinked product was improved. All the data prove that the crosslinked PES-OAllyl could be a potential solvent-resistance high-performance material.     

Global primary blast injury: a rat model

Blast wave injury from bombs cause a unique but poorly understood spectrum of injuries. Previous blast wave models involved high energy explosives detonated in an open field without the sophisticated monitoring of laboratory equipment. We characterized a rodent model that produces a global blast injury in a safe laboratory environment. Male rats, prospectively randomized to four groups of ten, were anesthetized and subjected to a blast at 2.0 cm, 2.5 cm, or 3.5 cm from the blast nozzle. The control group received no blast. Intensity of the blast (80-120 psi peak pressure, 1-2 msec duration) was controlled by varying the distance of the blast wave generator to the rat. The rats were monitored for three hours following the blast and then euthanized. Bradycardia was an immediate but transient response to blast injury. Mean arterial pressure was bimodal with severe hypotension occurring immediately after the blast and, again, two to three hours later. The characteristic injuries from a blast wave, such as pulmonary hemorrhage with increased lung weight, intestinal serosal hemorrhage, and hemoperitoneum, were found in the rats subjected to the blast pressure wave. In conclusion, our rodent model accurately reproduces the clinical spectrum of injuries seen in blast victims and will provide a powerful tool for studying the pathophysiology and potential treatments of bomb blast victims.