VEGF expression in hepatectomized tumor-bearing mice.

The experiments were designed in order to study the VEGF expression in intact (group I), hepatectomized (group II), and hepatectomized-tumor bearing mice (group III) throughout one complete circadian time span. Adult male mice were used for the VEGF expression study. The statistical analysis was performed using analysis of variance (ANOVA). The results showed statistical differences in the VEGF expression between groups I and II, but the most significant differences were found between groups I and III. In conclusion, these expressions have a circadian rhythm in all groups; moreover, in group III, this expression was higher and appeared before than in the others.

Evaluation of angiogenesis with the expression of VEGF and CD34 in human non-small cell lung cancer.

Angiogenesis is an essential process in the progression of malignant tumors and the most potent angiogenic factor is the vascular endothelial growth factor (VEGF). On the other hand, the CD34 is an endothelial antigen that has been used to highlight the microvasculature vessel density (MVD) as a direct marker of the degree of neoangiogenesis. In the present study we report the VEGF expression and its relationship with MVD, measured by CD34, in two lineages of non-small cell lung cancer (NSCL): low differentiated adenocarcinomas and epidermoid carcinomas, in order to consider the possibility of using the correlation between both antibodies as a prognostic factor. Tumor sections were stained by immunohistochemistry for CD34 and VEGF. The results showed that the mean value of VEGF for adenocarcinoma was significantly higher than the one for epidermoid carcinoma (p < 0.001). However, the mean of MVD did not show significant differences between both types of tumors. The conventional factors taken into consideration (age over 60, sex, and presence of lymph nodes) was not significantly related to the angiogenic factors examined. In conclusion, we could affirm that CD34 is a better prognostic marker of neoangiogenesis in NSCLC, because both types of tumors have the same clinical prognosis, and so we expected the same behaviour from both markers.

DNA synthesis in tongue keratinocytes of hepatectomized and tumor-bearing mice.

Tongue keratinocytes have high S-phase and mitotic indices with evident circadian variation. Transplanted tumors modify the intensity and temporal structure of the S-phase index in cell populations in tumor-bearing animals; also, partial hepatectomy changes the concentrations of substances involved in cellular proliferation, leading to compensatory liver hyperplasia. The aim of our study was to analyze the interaction between tumor growth and the liver regeneration that follows partial hepatectomy, and the effects of both these processes on lingual keratinocytes. We used 380 adult male mice divided into six groups: tumor-free and tumor-bearing mice without surgery, with sham hepatectomy, and with partial hepatectomy. Each group was divided into six subgroups, which were killed at 4-h intervals until a circadian cycle was completed (from 26 until 50h post-surgery in the operated animals). Each animal was injected with 5-bromodeoxyuridine (50mg/kg) 1h before it was killed, and tongue samples were obtained and processed for histology. The sections were placed on silanized slides and incubated with the primary antibody Bu 20a (1/100 dilution). The reaction was developed using diaminobenzidine and staining was detected visually. SIs were measured as the number of labeled nuclei per thousand cells. The mean+/-S.E. of each group was calculated. Differences among experimental groups were analyzed by ANOVA and the Student-Newman-Keuls Multiple Comparisons Test. The results show that the presence of a tumor alters the normal circadian curve of SI in lingual keratinocytes, irrespective of whether the mice underwent surgery. This finding has to be considered in drug treatments for neoplasms and in experiments related to growth.

Chronobiology of the proliferative events related to angiogenesis in mice liver regeneration after partial hepatectomy.

In liver regeneration the formation of new capillary blood vessels is a fundamental requirement for cellular proliferation. Vascular endothelial growth factor (VEGF) is involved in the events of angiogenesis, the mRNA of which is expressed in both hepatocytes and non-parenchymal cells. In this experimental design we try to establish if during liver regeneration in mouse, the expression of VEGF is produced before or after the hepatocytes proliferation. C3H/S adult male mice were divided in three groups in order to study: VEGF expression; S-phase index (SI); and mitotic activity (MA) of hepatocytes. The results that were analyzed by ANOVA, show that VEGF expression starts to increase 26 h after PH with a peak at 28 h. Furthermore, the DNA synthesis (DNAs) reaches maximal level 42 h after pH, meanwhile the MA of the hepatocytes shows an increase 8h after the DNAs peak. In conclusion, it could be argued that the chronobiology of the events related to liver regeneration in mice started with a release of VEGF by the hepatocytes, followed by its DNAs and mitosis.

Development of compensatory hepatic hyperplasia in mice carrying the hepatocellular carcinoma ES12a.

In a previous paper we reported that the presence of the hepatocellular carcinoma SS1K in host mice resulted in an earlier appearance of the hepatocyte mitotic peak during liver regeneration after a partial hepatectomy as well as in an increase in the amplitude of that mitotic wave. In the present work we analyse the effect of another hepatocellular carcinoma, the ES12a (HCES12a). Adult male mice of the C3H/S strain standardised for circadian-periodicity analysis, were used. One group received a subcutaneous graft of the HCES12a tumor, while another group served as control. Fifteen days later, all animals were submitted to a partial (70%) hepatectomy at 10:00 h and beginning at 16:00 h lots of between 5 and 9 host and control animals each were sacrificed at 4 h intervals until 16:00 h on the third day thereafter. All mice were injected with 2 microg/g colchicine 4 hrs before killing, and samples of livers were processed for hematoxylin-eosin staining. We determined the hepatocyte mitotic index for each animal and the mean value +/- the standard error of the mean for each lot. The peak of mitotic activity in the tumor-bearing animals took place four hours earlier than in control mice but the average values of hepatocytic mitotic activity were similar in both groups

Effect of partial and sham hepatectomy on the growth of a hepatocellular carcinoma.

We examined the effect of partial hepatectomy on the proliferation of hepatoma ES12a grafted into C3H/S mice compared to tumor growth in sham-hepatectomized controls. The animals were sacrificed every 4 hrs during three days from the 6th to the 78th h following each type of surgery. Unoperated tumor-bearing mice were likewise killed as controls, but only during one complete circadian period. All animals received 2 microg of colchicine per g of body weight intraperitoneally 4 hrs before decapitation. Measurement of mitotic indices in hematoxylineosin-stained tumor samples revealed a decrease in proliferation and a modification of the diurnal mitotic-activity profile in the hepatectomized and sham-operated animals from the first day after surgery. These differences persisted by the third postoperative day only in the hepatectomized animals. Thus, although surgical stress may initially affect tumor growth, the latter results must be the effect of the influence of the hepatic regeneration.

Circadian rhythm of DNA synthesis and mitotic activity in tongue keratinocytes.

Tongue keratinocytes have a high mitotic index (MI) with an evident circadian variation. Our study set out to compare and contrast two phases of the cell cycle: DNA synthesis (S-phase), with inmunocytochemical detection by bromodeoxyuridine (BrdU), and mitosis (M-phase), by the colchicine-arrest of metaphase method, exploring both the dorsal and ventral surfaces of the mouse tongue throughout a circadian period. Adult male mice standardized for light periodicity used for MI experiment were injected intraperitoneally with colchicine. Other animals were injected intraperitoneally with 5-BrdU for S-phase determination. Animals given both treatments were divided into six groups and killed at 4 h intervals until 20:00 h. Tongue samples were processed for histology and immuno-histochemistry. S and M indices were expressed as labelled nuclei or colchicine metaphases, respectively, per 1000 nuclei. Peak MI occurred at 12:00, with the minimum value at 20:00 on dorsal and ventral tongue surfaces. Peak S-phase was at 04:00, whereas the minimum value was at 16:00 for both surfaces. These results show that the proliferative activity of the tongue epithelium is of similar intensity and temporal distribution on both surfaces.

The relevance of hydroxyapatite and spongious titanium coatings in fixation of cementless stems. An experimental comparative study in rat femur employing histological and microangiographic techniques.

Pure titanium rods plasma-spray coated with hydroxyapatite (HA) or porous titanium (Ti) of controlled roughness were implanted bilaterally in the distal femur of Sprague-Dawley rats to compare the extent of bone growth on the two types of coating. The relevance of other factors, like mechanical stability and biological adaptation of the bone to the insertion of a foreign body implant, were investigated in femora which were over-reamed (absence of primary fit) or reamed without insertion of the rod. Continuous tetracycline labeling for the first 30 days and for the last 2 weeks in the 90-day group was performed; histological/histometric, fluorescence and microangiographic studies were carried out on serial sections of the implanted and control femora. In the group of stable implants, HA-coated rods showed 90% integration versus 53% with Ti-coated implants (P < 0.001); in over-reamed implants neither surface bone growth nor endosteal fixation occurred, and both types of rods were surrounded by a thick layer of connective tissue. The study documented early adhesion of osteoblasts and direct deposition of bone matrix on the substrate, while on spongious titanium osteogenesis was observed only in proximity to the surface. Remodeling of the reactive, primary bone to mature, lamellar bone took the form of a capsule surrounding the implants and radial bridges connecting the latter to the endosteal surface. The number, height and thickness of these bridges appeared to be the factors determining implant stability, rather than the extent of the bony capsule on the perimeter of the implant. Integration was a function not only of mechanical conditions and surface geometry, but also of the biological response of the whole bone to changes in the vascularization pattern. The reported phenomena can be seen more easily in experimental models involving small rodents because of their fast bone turnover and revascularization, but it is expected that they take place, even at a lower speed, in clinical situations like cementless stems of total hip replacement.

The effects of bone resorption inhibitors on the growth plate and proximal tibial metaphysis of rats: clinical implications.

This study investigated the effects of diphosphonates at scalar doses in a high bone turnover structure, namely, the proximal tibial metaphysis of rats. Arrest of bone modeling was represented by cylindrical-shaped metaphyses, increased height of the perichondrial bone bark, and persistence of primary metaphyseal trabeculae; these changes were dose-related. Higher doses of the inhibitors produced extension of the growth plate and arrest of the mineralization process. The dose-related dissociation between the effects on bone resorption and mineralization allows the therapeutic use of this class of drugs.

The effects of mechanical forces on bones and joints. Experimental study on the rat tail.

We have used an experimental model employing the bent tail of rats to investigate the effects of mechanical forces on bones and joints. Mechanical strain could be applied to the bones and joints of the tail without direct surgical exposure or the application of pins and wires. The intervertebral disc showed stretched annular lamellae on the convex side, while the annulus fibrosus on the concave side was pinched between the inner corners of the vertebral epiphysis. In young rats with an active growth plate, a transverse fissure appeared at the level of the hypertrophic cell layer or the primary metaphyseal trabecular zone. Metaphyseal and epiphyseal trabeculae on the compressed side were thicker and more dense than those of the distracted part of the vertebra. In growing animals, morphometric analysis of hemiepiphyseal and hemimetaphyseal areas, and the corresponding trabecular bone density, showed significant differences between the compressed and distracted sides. No differences were observed in adult rats. We found no significant differences in osteoclast number between compressed and distracted sides in either age group. Our results provide quantitative evidence of the working of 'Wolff's law'. The differences in trabecular density are examples of remodelling by osteoclasts and osteoblasts; our finding of no significant difference in osteoclast numbers between the hemiepiphyses in the experimental and control groups suggests that the response of living bone to altered strain is mediated by osteoblasts.

The reaction to nailing or cementing of the femur in rats. A microangiographic and fluorescence study.

Bone reaction to cement and to a cementless stem was studied in the rat femur with histological fluorescence and microangiographic techniques. Periosteal and endosteal apposition, and consequent remodelling, appeared as a reaction to reaming rather than caused by cement or a cementless stem. Every change in bone began with proliferation, progression and orientation of the vessels. Endosteal apposition was absent in cemented femurs because the entire medulla was occupied by the acrylic cement, but remodelling of the subendosteal cortex followed medullary revascularisation which was far advanced after 90 days. In cementless stems, endosteal apposition of primary woven bone and remodelling was the basis for bony ingrowth and anchorage through bony bridges. Our results suggest that the pattern of blood supply is relevant to the structural organisation of mature lamellar bone around the implant. Cemented stems have maximum anchorage and stability as soon as they are inserted, but this decreases with time as revascularisation occurs. Cementless stems can reach maximum integration later after insertion, and revascularisation is less critical because they usually do not fill the canal completely.

Pathology of disappearing bone disease: a case report with immunohistochemical study.

A case of disappearing bone disease of the proximal femur is reported with histopathological and immunohistochemical studies. There was a densely packed cellular tissue, positive to endothelial antibodies, in areas of massive bone destruction. A more differentiated vascular tissue was present where trabecular cancellous or cortical bone was preserved with only focal zones of accelerated bone remodelling. The self-limited course correlates well with two phases of evolution of the histopathological lesions with neoplastic-like proliferation of endothelial cells corresponding to the rapid and massive bone destruction, and a later differentiation of the cells in mature vascular structures, but still with accelerated bone resorption which is partly compensated by appositional activity.

Quantitative evaluation of the bone-metal interface in implants with two different surface roughnesses: an experimental study in rabbits.

Direct bone-metal contact is considered the ideal condition in order to obtain stability of orthopaedic non cemented implants. To acheive this result various implant shapes and surfaces were proposed.

Gait patterns after anterior cruciate ligament reconstruction.

The aim of this study is to analyse the changes in select gait parameters following anterior cruciate ligament (ACL) reconstruction. The study was performed on 15 subjects who underwent ACL reconstruction by the bone-patellar tendon-bone technique. Gait analysis was performed using the Elite three-dimensional (3D) optoelectronic system (BTS), a Kistler force platform and the Telemg telemetric electromyograph (BTS). Kinematic data were recorded for the principal lower limb joints (hip, knee and ankle). The examined muscles include vastus lateralis, rectus femoris, biceps femoris and semitendinosus. The results obtained from the operated subjects were compared with those of 10 untreated subjects and 5 subjects without ACL damage. In the operated subjects the knee joint angular values regained a normal flexion pattern for the injured limb during the stance phase. The analysis of joint moments shows: (a) sagittal plane: recovery of the knee flexion moment at loading response and during preswing; (b) frontal plane: recovery of the normal patterns for both hip and knee adduction-abduction moments during the entire stance phase. The examination of ground reaction forces reveals the recovery of frontal component features. The EMG traces show the normal biphasic pattern for the operated subjects as compared to the untreated subjects. The results suggest that the gait parameters shift towards normal value patterns.

The failed back syndrome.

In vertebral surgery we frequently observe patients who complain of lumbar pain at intervals of time which vary after surgery. The causes that may involve the persistence or the recurrence of these symptoms are many, after both decompression surgery, and spinal fusion. In order to favor a more rapid and accurate diagnosis, in our clinical we use a diagnostic algorithm based on the time at which the symptoms occurred. The most modern radiodiagnostic methods allow for a more accurate preoperative diagnosis and a more accurate diagnosis of the pathologies capable of perpetuating or re-accentuating lumbar pain postsurgery.

[A case of human subcutaneous dirofilariasis in the Turin hills]. / Un caso di dirofilariosi umana sottocutanea nella collina torinese.

The literature about human dirofilariasis is briefly reviewed with particular reference to the Italian epidemiology, and a case of subcutaneous localization of living Dirofilaria Repens is described. The infestation occurred on the Turin hills. The occurrence of dirofilariasis in a hilly zone is pointed out, because the most common occurrence in Piedmont has been reported from rice zones.

[Clinical study on a new cephalosporin: CGP 9000 (cefroxadine)]. / Ricerca clinica su una nuova cefalosporina: CGP 9000 (cefroxadina).

CGP 9000 (cefroxadine), a new cephalosporine derived from N-acyl-3-alkoxy-7-amino-3-cefem-4-carboxylic acid for exclusively oral use, has been experimented on 67 patients, 41 adults and 20 children. CGP 9000 appeared to possess good therapeutic activity, even in low doses: its rapid absorption and moderate sero-protein bond are a guarantee of an immediate and almost total bioavailability.

[Kinetic and clinical study of bacampicillin]. / Ricerca cinetica e clinica sulla bacampicillina.

A mean plasma concentration curve, with peaks within the first hour and moderate individual variations, was obtained after a single administration of 800 mg of bacampicillin to 5 healthy volunteers. An adequate antibiotic transfer from the circulation to the bronchial apparatus was demonstrated. Treatment of 34 patients suffering from ampicillin-sensitive bacterial diseases permitted clinical cure of all patients, without onset of side-effects. It is concluded that bacampicillin is preferable to oral ampicillin because of its kinetic and tolerance features.