Rabinovich Pavel, Prof., MD, PhD, Israel
Menjerizkaja Galina, PhD, Israel
Sabo Edmond, проф., MD, Israel; Axelrod Elizabed, Israel
Rabinovich Alexandr, MD, Israel
Gershman Diamant, Israel

Background It is well known that unicellular and primitive multi-cellular organisms stay alive for an indefinite time if their environment is regularly refreshed in the laboratory condition ^{1 2 3 4} . In such organisms every cell gets nutrients and eliminates the products of metabolism directly in its external environment. They are potentially immortal but are very sensitive to damage from external factors (sunlight, drying etc.^{5 6} ). The high animals have autonomic internal environment connective tissue. Sensitivity of such animals (including humans) to damage from external factors is lower than in primitive organisms but the appearance of connective tissue makes them mortal. The fluid circulating through the connective tissue supplies cells with nutritious, hormones and other humoral biological regulators and removes the metabolism products to the blood for elimination by kidneys with urine ^{7 8 9 10} . 
Therefore, cell activity depends on connective tissue permeability. The core of connective tissue are intercellular proteins named collagen, elastin and macromolecular compounds contain carbohydrate (basic substance). With aging, collagen becomes denser and loses water, elastin also becomes denser and collapse, in basic substance proteoglycans changes with glycoprotein. The connective tissue grows stout containing less water which makes it less permeable. The decrease of permeability impedes the fluid circulation and causes failure of the cell metabolism and cell functions regulation thus reducing the organism's functional activity that is the essence of the aging process.

Proposed Solution to Aging 
• We use methods of improving body cells environment, i.e the connective tissue. We aim at improving cell function and, therefore, reverse the aging process. This goal will be reached by artificial stimulation of old intercellular proteins destruction and simultaneously replacing it with new young proteins under the non-invasive control. 
• REJUVINATION prevents the increase of connective tissue density, which is the main cause of aging. By doing so, we provide sustainable functional ability to the organisms at the levels that are typical for younger organisms, therefore, reversing aging. 
• We propose a novel method for biological rejuvenation of by means of a mixture of enzymes and biological active chemical compounds named new rejuvenation agent . This agent leads to old intercellular proteins destruction with their replacement by the new “young” intercellular proteins while increasing permeability of the connective tissue and cells’ metabolism to “younger” level. 
The effect of our new Collagenolytic Agent (CLA) was studied in two ways:

1. The ability of the agent to provide the destruction of the old collagen and its chaging by new young collagen 
2. The ability of the agent to reverse the metabolism towards the younger level. 

• The collagen is the sole source of hydroxyproline. The increasing of urine hydroxyproline excretion is the sign of the collagen destruction intensification.
• The collagen destruction and restitution can be evaluated by hystochemical methods
• The rejuvenation effect on the metabolism was controlled by oxygen consumption measurement. The tissue oxygen consumption continuously decreases with age. So the oxygen consumption increasing after collagenolytic agent injection may be consider as sign of the rejuvenation.

The method was successfully tested on 29 rats in Biochemical Laboratory and the vivarium of the Hebrew University, Biotechnology Lab of the Lev College and Hadassa Ein Karem Medical School. 
The histochemical analysis was conducted in Alpert Medical School of Brown University The Molecular Pathology Core Aldrich Building (USA).

Results:
Tables 1 present the results of the experiments (20 rats) proving the rejuvenating effect of the suggested collagenolytic agent. The table 1 shows that collagenolytic cause considerable increasing of the hydroxyproline urinal excretion on the next day (in 1.5 times; p<0.01). 

Table 1. Results the measuring of the hydroxyproline urinary excretion and oxygen consumption.CL – Collagenolytic P – Significant level

The level of hydroxyprolin urinary excretion diminished almost to the initial at ten days after collagenolitic injection (in 1.1 times; p>0.05). 
Data of the histological and histochemical research were presented as table and as bar graphs (see below, Fig. 1, 2 and table 2). The distribution and density of the collagen and elastic fibers significantly differed between the control and the experimental groups. The control group presented a more abundant and compact dermal collagenous framework and a deeper involvement by the collagen fibers, as compared to the experimental group. In the later, the collagen fibers were less dense (more

sparsely distributed) and more superficially distributed then in the control group. The elastic fibers showed an inverse pattern where the experimental groups presented denser elastic framework and the fibrils were longer and thicker than the control group where elastic fibers were shorter, thinner and less densely distributed in the dermis. 
It is need to take notice of a dermis diameter. It is equal in control and experiment slides whereas deepest collagen diameter and deepest collagen percent in control were bigger then in experiment in 1.5 times (p<0.00053; Fig. 2; Table 2). Thus, the values of the cross section of the dermis in control and experiment were not different but the amount of the collagen is statistical significant lover in experiments. Preservation the initial thickness of the dermis in case of the collagen amount is decreased indicate that part of the old collagen is substitute for the new more hydrophilic good swelled collagen. 
Percent of the dermal area involved by collagen is less in treated animals than in control ones (Fig. 2). The increase of free space between collagen's fibers should increase the permeability of the connective tissue for the flow of the tissue liquid. It was astonishing that the injections of the collagenolytic agent cause the increasing of the elastin quantity in the connective tissue of the treated rates (in 1,6 time, p<0.0001; Fig. 2). 
Table 1 shows also significant increasing of the oxygen consumption during the all experiment and stable level of this index in all control. The level of oxygen consumption has risen at 1.4 time from initial value in experimental group of the rats while in control group of animals this index remain closely 4 ml/kg/10 minutes (p<0.001). It is necessary to emphasize that the increase of the urinary hydroxyproline elimination and increase of the oxygen consumption were in figure meaning the same in experimental and control groups of the rats. 
Table 1 shows also significant increasing of the oxygen consumption during the all experiment and stable level of this index in all control. The level of oxygen consumption has risen at 1.4 time from initial value in experimental group of the rats while in control group of animals this index remain closely 4 ml/kg/10 minutes (p<0.001).

Conclusion The first stage of the study shows that collagenolytic agent injections resulted in: 

• Substantial intensification in the old collagen decomposition.
• Substantial intensification in the new collagen and elastic fibers performing.
• Considerable increasing of space between collagen's fibers and permeability of the connective tissue
• Considerable increasing of oxygen consumption which is a sign of the animal’s metabolism rising the evidence of the process rejuvenation .
• Therefore, this stage of experimental testing demonstrates the rejuvenation effect.

Reference
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Получено от авторов 11 сентября 2008г.