Preoperative Rehabilitation Is Feasible in the Weeks Prior to Surgery and Significantly Improves Functional Performance.

BACKGROUND: Frailty is a multidimensional state of increased vulnerability. Frail patients are at increased risk for poor surgical outcomes. Prior research demonstrates that rehabilitation strategies deployed after surgery improve outcomes by building strength. OBJECTIVES: Examine the feasibility and impact of a novel, multi-faceted prehabilitation intervention for frail patients before surgery. DESIGN: Single arm clinical trial. SETTING: Veterans Affairs hospital. PARTICIPANTS: Patients preparing for major abdominal, urological, thoracic, or cardiac surgery with frailty identified as a Risk Analysis Index≥30. INTERVENTION: Prehabilitation started in a supervised setting to establish safety and then transitioned to home-based exercise with weekly telephone coaching by exercise physiologists. Prehabilitation included (a)strength and coordination training; (b)respiratory muscle training (IMT); (c)aerobic conditioning; and (d)nutritional coaching and supplementation. Prehabilitation length was tailored to the 4-6 week time lag typically preceding each participant's normally scheduled surgery. MEASUREMENTS: Functional performance and patient surveys were assessed at baseline, every other week during prehabilitation, and then 30 and 90 days after surgery. Within-person changes were estimated using linear mixed models. RESULTS: 43 patients completed baseline assessments; 36(84%) completed a median 5(range 3-10) weeks of prehabilitation before surgery; 32(74%) were retained through 90-day follow-up. Baseline function was relatively low. Exercise logs show participants completed 94% of supervised exercise, 78% of prescribed IMT and 74% of home-based exercise. Between baseline and day of surgery, timed-up-and-go decreased 2.3 seconds, gait speed increased 0.1 meters/second, six-minute walk test increased 41.7 meters, and the time to complete 5 chair rises decreased 1.6 seconds(all P≤0.007). Maximum and mean inspiratory and expiratory pressures increased 4.5, 7.3, 14.1 and 13.5 centimeters of water, respectively(all P≤0.041). CONCLUSIONS: Prehabilitation is feasible before major surgery and achieves clinically meaningful improvements in functional performance that may impact postoperative outcomes and recovery. These data support rationale for a larger trial powered to detect differences in postoperative outcomes.

Laminin fragment E8 mediates PC12 cell neurite outgrowth by binding to cell surface beta 1,4 galactosyltransferase.

A number of cell surface receptors bind to distinct laminin domains, thereby mediating laminin's diverse biological activities. Cell surface beta 1,4-galactosyltransferase (GalTase) functions as one of these laminin receptors, facilitating mesenchymal cell migration and PC12 cell neurite outgrowth on laminin. In this study, the GalTase binding site within laminin was identified as the E8 fragment by assaying purified fragments and by immunoprecipitating and immunoblotting galactosylated laminin using E8-reactive antibodies. Compared with intact laminin and other laminin fragments, E8 possessed the highest GalTase binding activity, using both membrane-bound and solubilized GalTase. More significantly, the neurite-promoting activity of fragment E8 was shown to be dependent upon its interaction with GalTase. Pregalactosylating purified E8 eliminated subsequent GalTase binding and consequently inhibited neurite initiation; parallel studies on laminin fragments E1-4 or E1 failed to affect neurite outgrowth. Furthermore, anti-GalTase IgG inhibited neurite initiation on purified E8 substrates; control IgG had no effect. These results localize the predominant GalTase binding domain in laminin to fragment E8 and demonstrate that the neurite-promoting activity of E8 is dependent upon its interaction with GalTase.

Embryonic neural retinal cell response to extracellular matrix proteins: developmental changes and effects of the cell substratum attachment antibody (CSAT).

Cell attachment and neurite outgrowth by embryonic neural retinal cells were measured in separate quantitative assays to define differences in substrate preference and to demonstrate developmentally regulated changes in cellular response to different extracellular matrix glycoproteins. Cells attached to laminin, fibronectin, and collagen IV in a concentration-dependent fashion, though fibronectin was less effective for attachment than the other two substrates. Neurite outgrowth was much more extensive on laminin than on fibronectin or collagen IV. These results suggest that different substrates have distinct effects on neuronal differentiation. Neural retinal cell attachment and neurite outgrowth were inhibited on all three substrates by two antibodies, cell substratum attachment antibody (CSAT) and JG22, which recognize a cell surface glycoprotein complex required for cell interactions with several extracellular matrix constituents. In addition, retinal cells grew neurites on substrates coated with the CSAT antibodies. These results suggest that cell surface molecules recognized by this antibody are directly involved in cell attachment and neurite extension. Neural retinal cells from embryos of different ages varied in their capacity to interact with extracellular matrix substrates. Cells of all ages, embryonic day 6 (E6) to E12, attached to collagen IV and CSAT antibody substrates. In contrast, cell attachment to laminin and fibronectin diminished with increasing embryonic age. Age-dependent differences were found in the profile of proteins precipitated by the CSAT antibody, raising the possibility that modifications of these proteins are responsible for the dramatic changes in substrate preference of retinal cells between E6 and E12.

Isolation and characterization of a laminin-binding protein from rat and chick muscle.

A major laminin-binding protein (LBP), distinct from previously described LBPs, has been isolated from chick and rat skeletal muscle (Mr 56,000 and 66,000, respectively). The purified LBPs from the two species were shown to be related antigenically and to have similar NH2-terminal amino acid sequences and total amino acid compositions. Protein blots using laminin and laminin fragments provided evidence that this LBP interacts with the major heparin-binding domain, E3, of laminin. Studies on the association of this LBP with muscle membrane fractions and reconstituted lipid vesicles indicate that this protein can interact with lipid bilayers and has properties of a peripheral, not an integral membrane protein. These properties are consistent with its amino acid sequence, determined from cDNAs (Clegg et al., 1988). Examination by light and electron microscopy of the LBP antigen distribution in skeletal muscle indicated that the protein is localized primarily extracellularly, near the extracellular matrix and myotube plasmalemma. While a form of this LBP has been identified in heart muscle, it is present at low or undetectable levels in other tissues examined by immunocytochemistry indicating that it is probably a muscle-specific protein. As this protein is localized extracellularly and can bind to both membranes and laminin, it may mediate myotube interactions with the extracellular matrix.

Amino acid sequence and distribution of mRNA encoding a major skeletal muscle laminin binding protein: an extracellular matrix-associated protein with an unusual COOH-terminal polyaspartate domain.

Two cDNAs encoding an abundant chicken muscle extracellular matrix (ECM)-associated laminin-binding protein (LBP) have been isolated and sequenced. The predicted primary amino acid sequence includes a probable signal peptide and a site for N-linked glycosylation, but lacks a hydrophobic segment long enough to span the membrane. The COOH terminus consists of an unusual repeat of 33 consecutive aspartate residues. Comparison with other sequences indicates that this protein is different from previously described LBPs and ECM receptors. RNA blot analysis of LBP gene expression showed that LBP mRNA was abundant in skeletal and heart muscle, but barely detectable in other tissues. Blots of chicken genomic DNA suggest that a single gene encodes this LBP. The amino acid sequence and mRNA distribution are consistent with the biochemical characterization described by Hall and co-workers (Hall, D. E., K. A. Frazer, B. C. Hahn, and L. F. Reichardt. 1988. J. Cell Biol. 107:687-697). These analyses indicate that LBP is an abundant ECM-associated muscle protein with an unusually high negative charge that interacts with both membranes and laminin, and has properties of a peripheral, not integral membrane protein. Taken together, our studies show that muscle LBP is a secreted, peripheral membrane protein with an unusual polyaspartate domain. Its laminin and membrane binding properties suggest that it may help mediate muscle cell interactions with the extracellular matrix. We propose the name "aspartactin" for this LBP.

The alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers mediate cell attachment to distinct sites on laminin.

This study was undertaken to determine the roles of individual alpha/beta 1 integrin heterodimers in promoting cellular interactions with the different attachment-promoting domains of laminin (LN). To do this, antibodies to the integrin beta 1 subunit or to specific integrin alpha subunits were tested for effects on cell attachment to LN, to elastase fragments E1-4 and E1, derived from the short arms and core of LN's cruciform structure, and to fragment E8 derived from the long arm of this structure. The human JAR choriocarcinoma cells used in this study attached to LN and to fragments E1 and E8. Attachment to E1-4 required a much higher substrate coating concentration, suggesting that it is a poor substrate for JAR cell attachment. The ability of cells to attach to different LN domains suggested the presence of more than one LN receptor. These multiple LN receptors were shown to be beta 1 integrin heterodimers because antibodies to the integrin beta 1 subunit inhibited attachment of JAR cells to LN and its three fragments. To identify the individual integrin alpha/beta 1 heterodimers that mediate interactions with these LN domains, mAbs specific for individual beta 1 heterodimers in human cells were used to study JAR cell interactions with LN and its fragments. An anti-alpha 6/beta 1-specific mAb, GoH3, virtually eliminated cell attachment to E8 and partially inhibited attachment to E1 and intact LN. Thus the major alpha 6/beta 1 attachment domain is present in fragment E8. An alpha 1/beta 1-specific mAb (S2G3) strongly inhibited cell attachment to collagen IV and partially inhibited JAR attachment to LN fragment E1. Thus, the alpha 1/beta 1 heterodimer is a dual receptor for collagen IV and LN, interacting with LN at a site in fragment E1. In combination, the anti-alpha 1- and anti-alpha 6-specific antibodies completely inhibited JAR cell attachment to LN and fragment E1. Thus, the alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers each function as LN receptors and act together to mediate the interactions of human JAR choriocarcinoma cells with LN.

A cell culture model of the blood-brain barrier.

Endothelial cells that make up brain capillaries and constitute the blood-brain barrier become different from peripheral endothelial cells in response to inductive factors found in the nervous system. We have established a cell culture model of the blood-brain barrier by treating brain endothelial cells with a combination of astrocyte-conditioned medium and agents that elevate intracellular cAMP. These cells form high resistance tight junctions and exhibit low rates of paracellular leakage and fluid-phase endocytosis. They also undergo a dramatic structural reorganization as they form tight junctions. Results from these studies suggest modes of manipulating the permeability of the blood-brain barrier, potentially providing the basis for increasing the penetration of drugs into the central nervous system.

A neuronal cell line (PC12) expresses two beta 1-class integrins-alpha 1 beta 1 and alpha 3 beta 1-that recognize different neurite outgrowth-promoting domains in laminin.

Integrins mediate neuronal process outgrowth on components of the ECM. Integrin alpha subunit-specific antibodies have been used to examine the roles of individual beta 1 integrins in attachment and neurite outgrowth by the neuronal cell line, PC12, in response to laminin and collagen. alpha 1 beta 1 and alpha 3 beta 1 were identified as the major beta 1 integrins expressed by PC12 cells. In functional assays, both alpha 1 beta 1 and alpha 3 beta 1 mediated PC12 cell interactions with laminin, whereas alpha 1 beta 1 alone mediated responses to collagen types I and IV. alpha 1 beta 1 and alpha 3 beta 1 were shown to recognize two different neurite-promoting sites in laminin: alpha 1 beta 1 interacted with the cross-region of laminin present in proteolytic fragments E1-4 and E1; alpha 3 beta 1 recognized a site in the long arm contained in laminin fragment E8. Thus, PC12 cells express two beta 1 integrins, which together function in attachment and neurite outgrowth on laminin and collagen. These integrins are candidates for mediating neurite outgrowth of sympathetic and other neurons in response to these ECM components.

Activation of iCaspase-9 in neovessels inhibits oral tumor progression.

Tumors of the oral cavity are highly vascularized malignancies. Disruption of neovascular networks was shown to limit the access of nutrients and oxygen to tumor cells and inhibit tumor progression. Here, we evaluated the effect of the activation of an artificial death switch (iCaspase-9) expressed in neovascular endothelial cells on the progression of oral tumors. We used biodegradable scaffolds to co-implant human dermal microvascular endothelial cells stably expressing iCaspase-9 (HDMEC-iCasp9) with oral cancer cells expressing luciferase (OSCC3-luc or UM-SCC-17B-luc) in immunodeficient mice. Alternatively, untransduced HDMEC were co-implanted with oral cancer cells, and a transcriptionaly targeted adenovirus (Ad-VEGFR2-iCasp-9) was injected locally to deliver iCaspase-9 to neovascular endothelial cells. In vivo bioluminescence demonstrated that tumor progression was inhibited, and immunohistochemistry showed that microvessel density was decreased, when iCaspase-9 was activated in tumor-associated microvessels. We conclude that activation of iCaspase-9 in neovascular endothelial cells is sufficient to inhibit the progression of xenografted oral tumors.

Rapid and quantitative assessment of cancer treatment response using in vivo bioluminescence imaging.

Current assessment of orthotopic tumor models in animals utilizes survival as the primary therapeutic end point. In vivo bioluminescence imaging (BLI) is a sensitive imaging modality that is rapid and accessible, and may comprise an ideal tool for evaluating antineoplastic therapies. Using human tumor cell lines constitutively expressing luciferase, the kinetics of tumor growth and response to therapy have been assessed in intraperitoneal, and subcutaneous, and intravascular cancer models. However, use of this approach for evaluating orthotopic tumor models has not been demonstrated. In this report, the ability of BLI to noninvasively quantitate the growth and therapeutic-induced cell kill of orthotopic rat brain tumors derived from 9L gliosarcoma cells genetically engineered to stably express firefly luciferase (9LLuc) was investigated. Intracerebral tumor burden was monitored over time by quantitation of photon emission and tumor volume using a cryogenically cooled CCD camera and magnetic resonance imaging (MRI), respectively. There was excellent correlation (r=0.91) between detected photons and tumor volume. A quantitative comparison of tumor cell kill determined from serial MRI volume measurements and BLI photon counts following 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment revealed that both imaging modalities yielded statistically similar cell kill values (P=.951). These results provide direct validation of BLI imaging as a powerful and quantitative tool for the assessment of antineoplastic therapies in living animals.

Immune responses to chlorpromazine in rats. Detection and relation to hepatotoxicity.

It has frequently been suggested that the jaundice which occurs in a small percentage of human patients following treatment with chlorpromazine is due to a hypersensitivity reaction. It has, however, proved impossible to obtain an animal model for this condition. We now show that oral administration of chlorpromazine at 25 mg/kg per day to Wistar albino rats results in formation of both humoral and secretory antibodies to chlorpromazine. We also demonstrate that the severity of the hepatic changes observed in chlorpromazine-fed animals (periportal glycogen loss and centrilobular fatty change) is enhanced by preimmunization of the rats via the gut-associated lymphoid tissue with a chlorpramizine-protein conjugate. There was, however, no correlation between the titre of either serum or biliary antibodies in individual animals and the degree of liver damage. Our results therefore suggest than an immune mechanism is indeed implicated in chlorpromazine toxicity but show clearly that toxic symptoms are not a simple consequence of the formation of anti-chlorpromazine antibodies.

Hydroxyproline metabolism by the rat kidney: distribution of renal enzymes of hydroxyproline catabolism and renal conversion of hydroxyproline to glycine and serine.

The metabolism of hydroxyproline by the rat kidney leads to the production of significant quantities of both glycine and serine. This process was observed in both the isolated perfused kidney and in isolated cortical tubule suspensions. The rate of hydroxyproline metabolism was increased in both preparations by the addition of alanine. The distribution of hydroxyproline oxidase, hydroxyoxoglutarate aldolase and alanine-glyoxalate transaminase were determined in detail. All three enzymes were found exclusively in the renal cortex where they were restricted to the mitochondria. Cortical tubule fractionation studies indicated that the enzymes are located in the proximal convoluted and proximal straight segments at the nephron. The results suggest that hydroxyproline degradation could contribute significantly to the renal synthesis of serine.

Effects of acute and sub-chronic administration of iron nitrilotriacetate in the rat.

Parenteral administration of iron nitrilotriacetate (FeNTA) to rats resulted in marked loss in body weight, and increases in liver/and kidney/body weight ratios. Fatalities, due to renal failure, depended on dosage and age of the animals, and were greater (70%) after a single large dose (12 mg iron) than after repeated smaller doses (30%). FeNTA administered subchronically gave rise to an increase in ethane exhalation, and to decreased liver glutathione peroxidase activity, and decreased cytochrome P-450 concentration and benzphetamine N-demethylase activity. It also resulted in severe renal tubular necrosis, with deposition of iron in the tubular cells and loss of brush border alkaline phosphatase activity, resulting in a dose-dependent diuresis, with increased urinary excretion of glucose, iron and lipid peroxidation products, and decreased urine creatinine concentration. NTA alone had none of these effects but slightly decreased the hepatic concentration of iron.

Time and dose study on the response of rats to the hypolipidaemic drug fenofibrate.

Groups of male Wistar albino rats were administered diets containing sufficient fenofibrate to ensure intakes of either 200, 60 or 13 mg/kg/day or sufficient clofibrate to ensure an intake of 400 mg/kg/day. Four rats from each experimental group and 6 control rats were killed, 3, 7, 14 and 28 days, 8, 12 and 20 weeks and 6, 9, 12 and 18 months after commencement of treatment. At all time points livers were subjected to histological, electron microscopic and biochemical examination, the other major abdominal organs were removed for histological examination. A more extensive necropsy was carried out on rats killed after 12 and 18 months. The major alterations were observed in the liver, although there were also morphological changes in the thyroid, pancreas and kidney after prolonged treatment. The hepatic changes followed a distinct time course. Within 24 h of offering diets containing the compounds to the rats there was accumulation of small droplets of lipid, induction of peroxisomal enzymes and of the specific cytochrome P-450 catalysing omega-hydroxylation of fatty acids and an increase in the number of mitotic figures. More slowly developing changes were loss from the centrilobular zone of fat, glycogen and of glucose 6-phosphatase activity. Here maximal changes were observed after 14 days of treatment. A still more slowly developing change was accumulation of enlarged lipid-loaded lysosomes, which was maximal at 26 weeks, accompanied by the development of lipofuscin bodies. Finally, in animals treated for 12 months or more there was evidence for increasing cell turnover as indicated by an increased number of mitotic figures, more dark cells and induction of serum alanine transaminase. The last 2 groups of changes were not observed in rats treated with 13 mg/kg/day of fenofibrate. In general the degree of change in rats treated with 400 mg/kg/day of clofibrate was similar to those found in rats treated with 60 mg/kg/day of fenofibrate.

A comparison of the protective effects of N-acetyl-cysteine and S-carboxymethylcysteine against paracetamol-induced hepatotoxicity.

The protective effect of the sulphur-containing amino acids N-acetyl-cysteine and S-carboxymethylcysteine against paracetamol-induced hepatotoxicity was evaluated in the hamster by biochemical and histological methods. Of the animals receiving paracetamol alone 25% died within 24 h following administration. All surviving animals showed acute hepatocellular injury and marked loss of cytochrome P-450 and hepatic mixed-function oxidase activities. Simultaneous administration of N-acetylcysteine decreased the mortality rate, partly prevented the paracetamol-induced liver damage and partly restored enzyme activities. Simultaneous administration of S-carboxymethylcysteine with paracetamol afforded no protection. Kidneys from all animals were histologically normal. Human liver microsomes and liver microsomes from 3-methylcholanthrene-pretreated hamsters metabolished paracetamol to intermediate(s) that bind covalently to microsomal proteins. The rate of covalent binding was inhibited markedly by N-acetylcysteine and to a lesser extent by S-carboxylmethylcysteine.

Lipid accumulation in the livers of chlorpromazine-treated rats does not induce peroxisome proliferation.

Treatment of rats with 25 mg/kg/day of the neuroleptic drug chlorpromazine for periods of 7, 28 or 90 days causes a slow accumulation of lipid in large droplets in centrilobular hepatocytes. There is little or no damage to hepatocytes as assessed by changes in glucose-6-phosphatase activity and by electron microscopy. Furthermore there is no indication of a change in peroxisomal beta-oxidation of fatty acids or in microsomal omega-oxidation of fatty acids. It is, therefore, clear that lipid accumulation in the liver does not automatically induce peroxisomal and microsomal fatty acid oxidising enzymes.

Comparison of the short-term hepatic effects of orally administered citral in Long Evans hooded and Wistar albino rats.

The short-term effects of citral on the liver have been studied in two strains of rat. Hepatomegaly was accompanied in citral-treated rats by an altered distribution of lipid and glycogen in the liver and peroxisome proliferation occurred in a manner reminiscent of that associated with some hypolipidaemic compounds. Specific biochemical markers supported the morphological changes in the peroxisomes. Cyanide-insensitive palmitoyl CoA oxidation showed, at the maximum, fourfold and threefold inductions in Wistar albino and Long Evans hooded rats, respectively. In addition, induction of cytochrome P-450 levels was greater in the Long Evans than in the Wistar rats, the maximal increases recorded being 81 and 27% respectively. A peroxisome-associated polypeptide of molecular weight 80,000 daltons (PPA-80) was induced, especially in Long Evans rats. No alterations in plasma triglycerides or total cholesterol were detected. The differential induction of the mixed-function oxidase system and the differential proliferation of peroxisomes in these two strains of rat suggest that citral may be metabolized differently in the two strains. The study indicates that peroxisomal and possibly also mitochondrial changes are involved in the action of citral on lipid metabolism.

The effect of megadose ascorbate on some haemopoietic factors in the young guinea pig.

The effect of oral administration of megadoses of ascorbate was studied on some haemopoietic factors in young apparently normal guinea pigs. Supplementation of 150 mg of the vitamin for 7 days resulted in significantly increased haematocrit value and haemoglobin concentration. Total and unsaturated iron binding capacity were also increased in association with elevated plasma iron concentration. The difference of these results from the control animals, however, was not statistically significant. The improved haematological status in guinea pigs by ascorbate treatment could be attributed to haemoconcentration rather than to an effect of iron metabolism.

Influence of avian aflatoxicosis on the synthesis of polyamines.

Effects were examined of inanition, dietary aflatoxin (2.5 mg/kg), and dietary supplements of threonine, lysine, and arginine on the activities of renal arginase and hepatic ornithine decarboxylase and on the accumulation of polyamines in liver and brain of 24 or 26-day-old broiler cockerels. Aflatoxicosis and inanition lowered the activity of renal arginase by 58 and 37%, respectively. Supplemental dietary threonine (.4%) did not suppress the activity of renal arginase, while fortification of diets of controls with lysine (.53%), but not diets containing aflatoxin, elevated the activity of renal arginase. Supplements of dietary lysine and/or arginine did not influence the hepatic content of putrescine but lowered the concentrations of spermidine and spermine. Aflatoxicosis, but not inanition, increased the activity of hepatic ornithine decarboxylase (ODC; 22-fold), increased hepatic concentrations of putrescine and spermidine, but decreased spermine concentrations. The elevation of hepatic ODC, putrescine, and the ratio of spermidine to spermine parallels the enlargement of the liver caused by aflatoxicosis. Cadaverine and putrescine were not detected in avian brain, while cerebral concentrations of spermidine and spermine were not altered by aflatoxin, inanition, or by supplements of dietary lysine, arginine, or both lysine and arginine.