Treatment of simple elbow dislocation using an immediate motion protocol.

The results of treatment after closed reduction of elbow dislocation vary. Twenty consecutive patients with closed posterior elbow dislocations were treated prospectively on a rapid motion, nonimmobilized functional regimen. This treatment protocol emphasizes immediate active range of motion under close supervision. No slings or splints were employed. Final range of motion averaged -4 degrees to 139 degrees. All patients attained final extension within 5 degrees of the contralateral side. Each patient achieved his final range of motion within an average of 19 days after reduction of the dislocation. Arm circumference returned to normal at an average of 6.5 days. There was one redislocation. After treatment, all patients met qualification for graduation from the U.S. Naval Academy and were able to pursue unrestricted athletic and career options. Our findings suggest that an aggressive immediate motion rehabilitation allows nearly full final elbow motion and an excellent functional outcome.

Progression of meniscal degenerative changes in college football players: evaluation with MR imaging.

Intrameniscal degenerative changes, presumably due to mild repetitive trauma, have been shown in many college and professional athletes, but it is uncertain over what period of time they can develop or significantly progress. To ascertain this period, the authors used magnetic resonance (MR) imaging to examine one knee in each of 20 players in the starting lineup of a major college football team before and after the season. Only asymptomatic knees (right, n = 10; left, n = 10) were examined; the images were reviewed blindly by one experienced observer without reference to the other examination. A significant progression existed in the grade of signal intensity shown in the menisci over the course of the season (P less than .001). Although this is a small study covering only 1 year, these preliminary results suggest that significant degeneration can occur in the menisci of asymptomatic players over a single season.

The effect of estrogen and tamoxifen on hepatocyte proliferation in vivo and in vitro.

We have previously shown that changes in estrogen-hepatocyte interaction occur during liver regeneration. Following 70% hepatectomy, estrogen levels in the blood were elevated, the number of estrogen receptors in the liver was increased and there was an active translocation of estrogen receptors from the cytosol to the nucleus. The injection of tamoxifen, an estrogen antagonist, inhibits hepatocyte proliferation following partial hepatectomy. The administration of 1 microgram tamoxifen per gm body weight at zero time or 6 hr after the operation resulted in a significant inhibition both of DNA synthesis and of the number of cells in mitosis. Injections of tamoxifen 12 hr or later after the operation had no effect. Concomitant injections of equimolar amounts of estrogen abolished the inhibition by tamoxifen. The effects of estrogen and tamoxifen were also tested on hepatocytes in primary culture. Estrogens in the presence of 5% normal rat serum stimulated hepatocyte DNA synthesis as determined by [3H]thymidine incorporation and the labeling index, whereas epidermal growth factor-induced DNA synthesis in the absence of normal rat serum was strongly inhibited. Tamoxifen, in contrast, inhibited DNA synthesis of hepatocytes in the presence of 5% normal rat serum and reversed the stimulatory effect of estrogen in the same system. Attempts to elucidate the mechanism of tamoxifen inhibition in vitro indicated that one effect of tamoxifen is to prevent the amiloride-sensitive Na+ influx necessary to initiate hepatocyte proliferation.

Excision of posterolateral talar dome lesions through a medial transmalleolar approach.

Posterolateral osteochondral fractures of the talus are rare. Although arthroscopy is becoming an increasingly important method of evaluating and treating lesions of the ankle, these techniques may not always be feasible, especially for posterolateral lesions. Classic treatment of displaced or symptomatic chronic lesions is excision, usually with a distal fibular osteotomy and turndown procedure. Subsequent removal of the syndesmosis screw is required. The surgical dissection of the distal fibula is extensive and devascularizing. An alternate technique for debriding posterolateral talar dome lesions through a medial transmalleolar approach is described. Exposure of the lateral talar dome is sufficient to allow debridement and curettage of the lesion. Anatomic rigid fixation of the medial malleolus allows for rapid healing of the osteotomy site and immediate ankle rehabilitation. For those ankle lesions that are not accessible to arthroscopy or an anterolateral arthrotomy, this approach is preferable to the distal fibular osteotomy and turndown.

Pharmacologic modulation of experimental postischemic hepatic function.

The present study evaluated and compared the effects of SRI 63-441, a potent platelet activating factor antagonist, superoxide dismutase (SOD), an oxygen free radical scavenger, and ibuprofen, a cyclooxygenase inhibitor on hepatic function after 90 minutes of warm ischemia. After warm ischemia, livers were harvested and underwent 90 minutes of warm, oxygenated, sanguinous perfusion on an isolated liver perfusion apparatus. Pretreatment of donor animals with 20 mg/kg intravenous (I.V.) SRI 63-441 5 minutes before induction of total hepatic ischemia resulted in significantly increased bile production, a significant decrease in transaminase release, and a higher tissue adenosine triphosphate (ATP) content when compared with ischemic nontreated controls. SOD resulted in improved bile production and decreased transaminase liberation only when present in the perfusate at the time of in vitro reperfusion. Ibuprofen did not improve postischemic hepatic function in this model. Electron microscopy revealed patchy hepatocellular vacuolization with an intact sinusoidal endothelium in all ischemic livers. However, the degree of damage was less severe in the livers from those rats pretreated with 20 mg/kg SRI 63-441. This study demonstrates that SRI 63-441 pretreatment significantly reduces hepatic warm ischemic injury, and in the present model, appears superior to two other agents that have been advanced in the treatment of ischemic injury. The use of such agents singly or in combinations have important implications as regards gaining a better understanding of the basic mechanisms in organ ischemia, and moreover, for therapeutic applications in organ ischemia and preservation.

Response of cultured hepatocytes to a hepatomitogen after initiation by conditioned medium or other factors.

Injection of a substantially purified hepatomitogen into recipient rats that had 40% of their liver removed resulted in a significant stimulation of hepatic DNA synthesis as determined by the labeling index and the mitotic index. Normal or sham-operated rats did not respond to the injection of the mitogen. The extraction and partial purification of this hepatomitogen have previously been reported (A. Francavilla et al., Cancer Res., 47:5600-5605, 1987). Addition of the factor to an epithelial-like liver-derived cell line in culture (clone 9) or to a hepatoma cell line (HTC-SR) resulted in a dose-dependent stimulation of DNA synthesis. Hepatocytes in primary culture, on the other hand, were not stimulated by the addition of the factor. However, when the mitogen was added to hepatocytes in primary culture, together with conditioned medium, obtained from the responsive cell lines, a significant stimulation of DNA synthesis could be demonstrated in hepatocytes in culture. The stimulation was dose dependent with respect to the mitogen, was abolished by 10 mM hydroxyurea, and was independent of epidermal growth factor. The conditioned medium could be replaced by a protein factor extracted from the two cell lines as previously reported (P. Ove et al., J. Cell. Physiol., 131: 165-174, 1987). It appears that a cofactor is provided by the conditioned medium or by the cell extract, enabling the hepatomitogen to act on hepatocytes in primary culture.

Improved hepatic function in the 24-hour preserved rat liver with UW-lactobionate solution and SRI 63-441.

The present study compares rat liver preservation for 9, 12, and 24 h in the standard Eurocollins solution with preservation for the same time periods in the new UW-lactobionate solution. Pharmacologic manipulation with a potent platelet-activating factor antagonist, SRI 63-441, was also evaluated. After cold storage in each of the test solutions, the livers underwent 90 min of warm, oxygenated, sanguinous perfusion. A significant increase in liver weight was noted in Eurocollins-stored versus UW-lactobionate-stored livers. After 90 min of perfusion, livers preserved in UW-lactobionate produced significantly more bile and liberated significantly less glucose and transaminases when compared with Eurocollins-stored livers. Significant augmentation of bile production was observed when donor animals were pretreated with SRI 63-441 and the livers were then stored in UW-lactobionate for 24 h. Eurocollins-stored livers demonstrated increased hepatocyte vacuolization and endothelial disruption when compared with UW-lactobionate-stored livers after 12 and 24 h of preservation. This study demonstrates the superiority of UW-lactobionate solution in liver preservation and suggests that SRI 63-441 may be beneficial in the further reduction of cold ischemic injury.

Extraction and partial purification of a hepatic stimulatory substance in rats, mice, and dogs.

A factor has been isolated from weanling rat liver which stimulates in vivo hepatic DNA synthesis in a dose dependent manner when injected into 40% hepatectomized rats. The factor has been partially purified by successive steps, involving ethanol precipitation, ultrafiltration through an Amicon PM 30 membrane, and finally fast protein liquid chromatography, resulting in a 38,000-fold increase in specific activity over that in the original cytosol. The factor contains a few bands in the molecular weight range of 14,000-50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Active fractions from fast protein liquid chromatography (F150), when injected into 40% hepatectomized rats, increased hepatic DNA synthesis 3-fold over the background stimulation due to the hepatectomy. The response was dose dependent over a range from 1.76 micrograms to 6.8 micrograms per 200-g (body weight) rat. Mitotic and labeling indexes confirmed that F150 stimulates both replicative DNA synthesis and cell proliferation. The factor is heat and neuraminidase resistant, trypsin sensitive, organ specific, but not species specific.

Different response to epidermal growth factor of hepatocytes in cultures isolated from male or female rat liver. Inhibitor effect of estrogen on binding and mitogenic effect of epidermal growth factor.

Deoxyribonucleic acid (DNA) synthesis in hepatocytes isolated from the livers of male and female rats has been compared in monolayer culture. Plating efficiency, DNA and protein content, viability, and morphologic appearance were the same in cultures prepared with hepatocytes isolated from male or female rats. Epidermal growth factor (EGF)-induced DNA synthesis was significantly higher in hepatocytes from male rats than in hepatocytes from female rats. This was the case whether hepatocytes were isolated from normal or partially hepatectomized male or female rats. Hepatocytes isolated from regenerating liver synthesize more DNA than those isolated from normal liver in response to EGF. This increased response to EGF in hepatocytes derived from regenerating liver was relatively the same for male- and female-derived hepatocytes, but the magnitude of the response was considerably higher in male-derived hepatocytes. In contrast, in vivo DNA synthesis in the liver remnant after partial hepatectomy was similar in male and female rats if measured 24 h after the operation. A comparison of EGF binding to male- and female-derived hepatocytes maintained in primary culture indicated a lower number of high-affinity receptors for EGF in the female hepatocytes. The addition of estrogen to primary cultures of hepatocytes isolated from male rats inhibited EGF binding as well as EGF-induced DNA synthesis. Our studies show significant differences in DNA synthesis in response to EGF when male and female hepatocytes are compared in primary culture. The regenerative response after partial hepatectomy, on the other hand, was the same in male and female rats. Thus, our studies indicate that the sex of the donor, rat is important when hepatocytes in culture are used for a variety of studies, such as hepatocyte metabolism, induction and control of DNA synthesis, and hepatocarcinogenesis. In addition, our results indicate that caution is advised when inferences are made from in vitro findings for in vivo conditions.

Isolation of an autocrine growth factor from hepatoma HTC-SR cells.

A growth factor has been isolated from HTC-SR rat hepatoma tissue culture cells which specifically stimulates DNA synthesis and cell proliferation of the HTC cells that produce it. The factor can be isolated from HTC cell conditioned medium or from an HTC cell extract. This autocrine factor has been purified 640-fold from a postmicrosomal supernatant by successive steps, involving ethanol precipitation, heating at 80 degrees C for 10 min, chromatography on a DEAE Bio-Gel A column, and chromatography on a heparin-sepharose affinity column. The major peak of activity eluted from the heparin column migrates as a single band on SDS-PAGE with an apparent Mr of 60,000. The factor is resistant to acid, heat, and neuraminidase but sensitive to trypsin, papain, and protease. The autocrine nature of the factor is indicated by the finding that several other types of cells do not respond with increased DNA synthesis. Mouse L-cells, BHK cells, Novikoff hepatoma cells, hepatocytes in primary culture, and an epithelial-like rat liver-derived cell line (Clone 9) were tested, and none of the cells could be stimulated. Small amounts of the factor could be extracted from the Clone 9 cells, however. This material had the same physical and purification properties as the factor extracted from HTC cells, but it did not stimulate DNA synthesis in Clone 9 cells, only in HTC cells. Addition of the factor resulted in an almost immediate stimulation of DNA synthesis in a proliferating HTC cell population. When the factor was added together with [3H]thymidine for 2 h, a significant stimulation of DNA synthesis was observed, provided the addition was made between 18 and 48 h after the cells had been plated. Autoradiographic studies indicated that the factor both accelerates DNA synthesis in cells already making DNA and increases the number of cells entering the S period. The stimulation of DNA synthesis was completely inhibited by 10 mM hydroxyurea, whether the factor was present for 2, 24, or 48 h in the culture. A significant increase in cell number due to addition of the factor was also observed. This accelerated proliferation was detectable only after the cells had been in culture for at least 48 h with the factor present.

Epidermal growth factor and proliferation in rat hepatocytes in primary culture isolated at different times after partial hepatectomy.

Primary hepatocyte cultures have been prepared from normal adult rat liver and from rat liver at 4, 8, 12, 24, and 48 h following partial hepatectomy (removal of 70% of the liver). Cells were maintained in minimal essential medium alone or supplemented with hormones. Comparing DNA synthesis in normal adult rat hepatocytes with DNA synthesis in hepatocytes isolated from regenerating livers, we found with minimal essential medium alone little DNA synthesis in normal adult rat hepatocytes and in hepatocytes isolated 4, 8, or 12 h after 70% hepatectomy. In hepatocytes isolated 24 h after partial hepatectomy, however, the incorporation of [3H]thymidine was 3 times the rate of normal hepatocytes. The addition of insulin to minimal essential medium had minimal effect on DNA synthesis in all hepatocytes. Addition of epidermal growth factor alone or in combination with insulin resulted in a dramatic increase in DNA synthesis in hepatocytes from regenerating rat liver. Increased incorporation was detectable as early as 4 h after partial hepatectomy and reached a maximum at 24 h after the operation. Results obtained with [3H]thymidine incorporation were confirmed by autoradiography and by direct DNA determinations in hepatocyte cultures. Epidermal growth factor binding to the hepatocytes was determined and agreed with previously reported binding studies. Binding of epidermal growth factor in hepatocytes isolated at 4 h after partial hepatectomy was the same as in normal hepatocytes but was undetectable in hepatocytes isolated from rats at 12 and 24 h after partial hepatectomy.

A comparison of DNA repair synthesis in primary hepatocytes from young and old rats.

DNA repair synthesis has been compared in primary hepatocyte cultures obtained from 3-month-old and 16-20-month-old rats. Several morphological and metabolic characteristics were determined to assure cultures of comparable quality. DNA damage was induced by the addition of bleomycin or the exposure of the culture to UV irradiation. DNA repair (unscheduled DNA synthesis) was determined by measuring [3H]thymidine incorporation. After UV irradiation, there was almost twice as much [3H]thymidine incorporation in cells obtained from young rats as in those obtained from old rats. Equal amounts of bleomycin resulted in substantially greater damage to DNA in cells from old rats than from young rats. For equal amounts of DNA damage there was again diminished [3H]thymidine incorporation in cells obtained from old rats. Finally equal amounts of bleomycin resulted in equal damage to DNA when the bleomycin was added to isolated rat liver nuclei from young or old rats. Bleomycin treated nuclei from young rats incorporated substantially more [3H]thymidine triphosphate (TTP) than bleomycin treated nuclei from old rats. The results indicate that hepatocytes from old rats are much more susceptible to bleomycin than hepatocytes from young rats and that the capacity for DNA repair synthesis is impaired in hepatocytes from old rats.

Endogenous hepatic growth-modulating factors and effects of a choline-devoid diet and of phenobarbital on hepatocarcinogenesis in the rat.

The activities of an endogenous inhibitor and of a stimulator of cell proliferation were assayed in the livers of sham-operated (SO) or partially hepatectomized (PH) adult rats; rats fed a choline-supplemented (CS) or a choline-devoid (CD) diet; the same diets followed by acute CCl4 intoxication; the same diets supplemented with phenobarbital (PHB); or a CD diet containing DL-ethionine (ETH). The inhibitor and the stimulator were semipurified by fractional ethanol precipitation of a liver cytosolic fraction, and their activities were assessed by means of bioassays in vitro. The livers of SO rats and of rats fed the CS diet contained only inhibitor activity. Following PH, a CD diet, or CCl4 intoxication the inhibitor activity was suppressed, and there was a simultaneous appearance of a stimulator activity. Thus, PH, a CD diet, and CCl4 intoxication cause similar cellular (loss and regeneration) and humoral-homeostatic changes in adult rat livers. We propose that these changes constitute a basic attribute of the mechanism whereby the three conditions affect similarly hepatocarcinogenesis in the rat, especially in the case of a CD diet, because the changes it induces are chronic rather than acute. PHB, another promoter of chemical hepatocarcinogenesis, affected neither the inhibitor nor the stimulator activity. Thus, PHB seems to be acting by a different mechanism than that of the other three agents. ETH did not modify the shift in the balance of the growth-modulating factors induced by a plain CD diet. This shift may account for the marked stimulation of carcinogen-induced oval cell proliferation exerted by a CD diet. The significance of these results is discussed in the context of known effects of a CD diet and of PHB on hepatocarcinogenesis in rats.

Discordance between glucokinase activity and insulin and glucagon receptor changes occurring during liver regeneration in the rat.

During regeneration of rat livers following 70% hepatectomy, insulin binding sites on hepatocyte plasma membranes are increased after 24-48 hours, glucagon binding sites are reduced on days 2-8, and the resultant insulin/glucagon binding ratio is markedly increased. An apparent paradox was the finding of a depression of the activity of an insulin associated enzyme, glucokinase, at a time when the number of insulin binding sites was increased.

Estrogen binding protein activity in Morris hepatoma 7777 compared with normal rat liver.

Estrogen binding protein activities were determined in the cytosol from adult male Buffalo rat liver and Morris hepatoma 7777. Estrogen receptors were prepared using the protamine sulfate precipitation technique of Chamness. The ability of various unlabeled steroids competing with [3H]estradiol was examined to establish the binding specificity. Estradiol binding in Morris hepatoma 7777 cytosol was greatly decreased compared with that present in hepatic cytosol prepared from normal rat liver. The receptor concentration expressed as femtomoles per milligram of cytoplasmic protein was 31.1 +/- 2.9 SD for normal rat liver and 0.41 +/- 0.88 SD for the hepatoma. Gel filtration chromatography revealed the presence of an estrogen binder in hepatoma cytosol which was not present in either normal liver or in the protamine sulfate precipitates of hepatoma cytosol. The molecular weight, binding specificity, and precipitation of this protein by specific antiserum suggests that it is alpha-fetoprotein.

Induction of hepatocyte stimulating activity by T3 and appearance of the activity despite inhibition of DNA synthesis by adriamycin.

A hepatocyte stimulating activity (HSA) has been extracted from rats that had received an injection of a pharmacological dose of T3 20 hours earlier. The injection of HSA from T3-treated rats into different recipient rats that had previously had 40% of their liver removed resulted in a significant increase in hepatic DNA synthesis. The injection of saline or HSA from normal rat liver had little or no effect on hepatic DNA synthesis in recipient rats. HSA from the T3-treated rats also stimulated DNA synthesis in Novikoff hepatoma cells and primary hepatocytes in culture, and in isolated normal rat liver nuclei in a nuclear incorporating system. In further experiments in which the increased DNA synthesis that follows partial hepatectomy was blocked by adriamycin, HSA appeared in these non-regenerating livers. This latter observation had indicated that the development of HSA is not merely an accompaniment of DNA synthesis.