An experimental model for chronic lymphedema

Although a multitude of operations exist for the treatment of lymphedema, none is highly successful. An experimental model that reliably and easily produces chronic lymphedema in an extremity would be useful to study treatments in a controlled and comparative manner and would enhance our understanding of the physiology and treatment of lymphedema. Many models that simulate clinical lymphedema have been described, but they suffer from cumbersome protocols, high laboratory costs, and an inconsistent yield of permanent lymphedema. We describe an experimental model for chronic lymphedema in the lower extremity of the rat that creates a lymphatic block in the groin induced by radiation treatment and one operation--surgical division of the superficial and deep lymphatics. All animals develop stable chronic lymphedema of the lower extremity within days of operation, with swelling that persists for at least 9 months. A mortality rate of 8 percent was associated with this technique. Methods for quantification of limb swelling are described, as is analysis of the lymphatic block by lymphoscintigraphic imaging of lymph channels and nodes. This model has the advantages of simplicity of technique, cost-effective use of rodent subjects, reproducibility of lymphedema, and quantification of results.     

The lumbrical fat graft: a replacement for lost upper eyelid fat

Human eyelids seem to age earlier and more rapidly than do other regions of the face, making blepharoplasty (either age or familially indicated) an operation often performed earlier than other remedial procedures for facial aging. The traditional and more doctrinaire approach to upper blepharoplasty in which fat and orbicularis muscle are excised has led to a type of iatrogenia that often leaves the upper (and lower) eyelids hollowed and vacant, before their time. The notion of upper blepharoplasty as a simple operation is, therefore, to be questioned. The idea of normal and obligatory asymmetry as a key to recognizability is brought into relief when contemplating blepharoplasty. In questioning the judgments by which upper palpebral fat (and orbicularis muscle) is excised, the author suggests both a remedy-the lumbrical fat graft-and the need for a heightened awareness of the synergy such excisions make with the age-related palpebral fat atrophies. This is a report of 35 cases (70 eyelids), over a 24-month period, during which the current technique was used. The technique is relatively simple, the anatomy well known, the learning slope shallow, the results gratifying, and the complications and problems few and benign.     

An animal paired crossover ePTFE arteriovenous graft model

Purpose

Previously, we developed a porcine model for Arterio Venous Graft (AVG) failure to allow assessment of new access strategies. This model was limited concerning graft length. In the present technical report, we describe a modification of our model allowing the assessment of long AVGs.

Technique

In 4 pigs, AVGs of 15 cm length were created bilaterally in a cross-over fashion between the carotid artery and the contralateral jugular vein. Two days (2 pigs) and two weeks (2 pigs) after AV shunting, graft patency was evaluated by angiography, showing all four grafts to be patent, with no sign of angiographic or macroscopic narrowing at the anastomoses sites.

Conclusions

In this modified pig AVG failure model, implantation of a bilateral cross-over long AVG is a feasible approach. The present model offers a suitable tool to study local interventions or compare various long graft designs aimed at improvement of AVG patency.     

An experimental model of pulmonary aspergillosis

The model of experimental chronic pulmonary aspergillosis has been obtained in mice and guinea pigs by the intrapulmonary infection of the animals with the suspension of Aspergillus mycelium and spores in complete Freund's adjuvant. Such method of infection has made it possible to produce chronic local mycotic process. Morphologically, focal inflammatory changes of the lung tissue with the subsequent formation of a multitude of small abscesses in these areas have been observed as characteristic manifestations of aspergillosis.     

An experimental model of tardive dyskinesias

Rats treated continually and chronically with trifluoperazine (ca 3 mg/kg/day) for six months initially developed mild catalepsy and an inhibition of spontaneous locomotor activity; both effects disappeared by three months. An initial increase in dopamine turnover (as measured by levels of homovanillic acid and dihydroxyphenylacetic acid) also disappeared by three months. Apomorphine-induced stereotypy was completely inhibited in drug-treated animals at two weeks, but progressively returned to normal after three months of drug intake. An exaggerated response to apomorphine developed in animals after six months of drug administration. Inhibition of striatal dopamine-stimulated adenylate cyclase found during the first month of drug intake was reversed at three months, a trend exaggerated after continuous drug administration for six months. Specific striatal ³H-spiperone binding affinity decreased acutely, but was increased after six months drug intake; no change in number of receptor sites occurred.These changes suggest that at least striatal dopamine receptors may become “supersensitive” during chronic neuroleptic treatment.     

Preparation of pre-confluent retinal cells increases graft viability in vitro and in vivo: a mouse model

Purpose

Graft failure remains an obstacle to experimental subretinal cell transplantation. A key step is preparing a viable graft, as high levels of necrosis and apoptosis increase the risk of graft failure. Retinal grafts are commonly harvested from cell cultures. We termed the graft preparation procedure “transplant conditions” (TC). We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigment epithelial (RPE) cell line, DH01.

Methods

Cell viability was assessed by trypan blue exclusion. Levels of apoptosis and necrosis in vitro were determined by flow cytometry for annexin V and propidium iodide and Western blot analysis for the pro- and cleaved forms of caspases 3 and 7. Graft viability in vivo was established by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunolabeling of subretinal allografts.

Results

Pre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%±0.8% vs. 13.1%±0.9%, p<0.01). Cell viability in either group was not altered significantly following TC. Caspases 3 and 7 were not altered by levels of confluence or following TC. Pre-confluent cultures had low levels of apoptosis/necrosis (5.6%±1.1%) that did not increase following TC (4.8%±0.5%). However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%±0.9%). Allografts prepared from post-confluent cultures had significantly more TUNEL-positive cells 3 hours post-operatively than grafts of pre-confluent cells (12.7%±3.1% vs. 4.5%±1.4%, p<0.001). Subretinal grafts of post-confluent cells also had significantly higher rates of cleaved caspase 3 than pre-confluent grafts (20.2%±4.3% vs. 7.8%±1.8%, p<0.001).

Conclusion

Pre-confluent cells should be used to maximize graft cell viability.     

An experimental model of DIC syndrome

Disseminated intravascular coagulation syndrome (DIC-syndrome) is experimentally reproduced by introduction of 30% aqueous solution "Desoxon-3" (2800-300 mg/kg) into the organism of laboratory animals (rabbits). The preparation was perorally introduced once on an empty stomach for 1 min. Biochemical and pathomorphological studies testify to the development of the typical DIC-syndrome in rabbits. It is concluded that the model suggested makes it possible to thoroughly study genesis, course, consequences of the development and preventive measures of disseminated intravascular blood coagulation, a complex pathological process.     

A coupling process for improving purity of bacterial minicells by holin/lysin

Drug and gene delivery systems using bacterial minicells have attracted much attention. Here we attempted to enhance the yield and purity of the minicells using a novel method in which autolysin induced in actively metabolizing parent cells led to autolysis. A two-step protocol coupling an autolysin (holin/lysin) step with a conventional centrifugation step achieved a purity of the minicells similar to that resulting from successive six- or seven-step methods.     

A quantitative genetics model for viability selection

Viability selection will change gene frequencies of loci controlling fitness. Consequently, the frequencies of marker loci linked to the viability loci will also change. In genetic mapping, the change of marker allelic frequencies is reflected by the departure from Mendelian segregation ratio. The non-Mendelian segregation of markers has been used to map viability loci along the genome. However, current methods have not been able to detect the amount of selection (s) and the degree of dominance (h) simultaneously. We developed a method to detect both s and h using an F2 mating design under the classical fitness model. We also developed a quantitative genetics model for viability selection by proposing a continuous liability controlling the viability of individuals. With the liability model, mapping viability loci has been formulated as mapping quantitative trait loci. As a result, nongenetic systematic environmental effects can be easily incorporated into the model and subsequently separated from the genetic effects of the viability loci. The quantitative genetic model has been verified with a series of Monte Carlo simulation experiments.     

An experimental model for studying radiation-induced suppression of antitumor resistance

In experiments on mice (CBA × C57Bl)F₁ an experimental model is developed for studying the effect of radiation on antitumor resistance. Adequate ways are offered for statistical processing of the data on enhancement of tumor growth in the irradiated organism. The validated model is a solid tumor developing after inoculation of a mouse with 1000 cells of Ehrlich ascites tumor into a hind shin, combined with suppression of antitumor resistance upon a 6-Gy (¹³⁷Cs) whole-body exposure. The recommended parameters for assessing tumor growth are: share of animals with tumors by day 30 after inoculation, length of the latent period, probability of tumor development, and tumor volume by day 30. For data processing, besides the common Fisher, χ² and Mann-Whitney tests, we recommend the Kaplan-Meier method and Log-rank. The proposed model can be a reliable tool in solving problems at the junction of radiobiology, oncology, immunology, pharmacology, etc.