Main Research Findings

1) The study confirms that cortagen restores neural functioning by eliciting a stimulatory effect on injured nerve fibers with decreased regeneration capacity.

2) Administration of cortagen was found to increase growth rate and conduction velocity of regenerating sciatic nerve fibers subjected to transection and suturing.

3) Treatment with peptides cortagen and epitalon results in decreased accumulation of lipid peroxidase byproducts and reduced oxidative protein modification.

 

Selected Data

1) The research team of Kolosova et al examined the ability of tetrapeptide, cortagen, to restore the functioning of injured nerve fibers. While large peptide molecules have been shown to improve development, growth, and regeneration of nerve tissue, there is lacking data regarding the efficacy of short chain peptides, such as cortagen, to elicit similar benefits. Previous research conducted by the team found that the peptide stimulates nerve regeneration in injured sciatic nerve fibers during the early stages of injury. For the purpose of this study the researchers shifted their focus to later stages of recovery and performed experimentation 5 months after the initial injury [1].

Throughout this study, 16 male Wister rats each weighing 200-250 grams each were utilized. The rats were randomized into an experimental treatment group and a control group that were intramuscularly injected with 10 ug/kg of cortagen or a saline solution, respectively, daily over 10 days. After 10 days, the animals were administered 50 mg/kg of ketamine and 15 mg/kg of rometar in order to perform operations under induced ketamine narcosis. The researchers then studied the sciatic-nerve functioning by transecting the nerve, suturing its ends with an epiperineural suture, and recording in vitro compound action potentials [1].

First, the vaseline bridge technique on a special ebonite platform was utilized to record the impulse activity of single fibers in order to study the functional properties of mechanoreceptors of the foot skin. From there, von Frey monofilaments were placed appropriately to determine the mechanical threshold for each of the single fiber receptors assessed. Evaluation of the receptors’ adaptive properties was also performed by applying a mechanical trapezoid stimulus of 20-50 ms at a three-threshold amplitude.

Finally, the researchers found that while preparing the sciatic nerve for in vitro testing, administration of cortagen resulted in a decrease in the neuroma located in the area of the suture. That being said, while assessing the conduction velocity of the action potential through the fragment of the regenerating sciatic nerve, conduction velocity was simultaneously observed at the area of the initial suture [1].

2) The research team of Turchaninova et al examined the ability of the tetrapeptide, cortagen, to improve functional recovery of injured sciatic nerve fibers. For the purpose of this study, male Wistar rats weighing 200-250 grams were utilized. The rats immediately underwent surgery and were anesthetized through an intramuscular injection of 45-50 mg/kg of ketamine and 10-20 mg/kg of rometar. In order to induce nerve damage the trunk of the sciatic nerve was transected proximally to the area where the cutaneous nerve branch separates from the common trunk and was further divided into the tibialis, peroneus, and suralis nerve branches. The nerve was then sutured under a microscope using 10/0 supramide thread, an atraumatic needle, and three epiperineural microsurgical sutures [2].

Following surgery, 10 ug/kg of cortagen was intramuscularly injected into the rats for 10 days starting immediately after surgery. The rats of the control group were treated following the same schedule but were injected with saline rather than an active compound. 4 weeks after surgery the compound action potential was evaluated in order to assess the effects of the tetrapeptide on the recovery of nerve conduction in regenerating nerves. In vitro testing of the compound action potential was completed using a special ebonite multielectrode platform that allowed the researchers to calculate the action potential parameters and locate the site of regeneration at a distance of up to 30 mm away from the stimulating electrodes [2].

The dissected sciatic nerve trunk was placed on the platform in order to ensure that the area of the suture coincided with the placement of the reference electrode. From there, the body temperature was warmed up by filling up the platform with warmed mineral oil. 0.2 msec rectangular pulses were generated using an ESU-2 electrical generator in order to stimulate the proximal end of the sciatic nerve trunk. The amplitude of the stimulus applied was 3 times the threshold value, resulting in a compound action potential that was recorded distally at the area of the initial suture. All recordings were amplified and fed into an oscilloscope for further monitoring. Based on the recordings the research team will measure latency of the action potential and restoration of conduction, as well as calculate the conduction velocity of the regenerating nerve [2].

3) Previous research conducted based on polypeptides epithalamin and cortexin has found that they are biological activity and have neuroprotective properties due to their antioxidant activity. Epithalamin elicits benefits by stimulating immunity and regulating both reproductive and neuroendocrine functioning, while cortexin stimulates regenerative processes in the central nervous system. That being said, the research team of Kozina et al utilized effective derivatives of epithalamin and cortexin, epitalon and cortagen, to examine the effects of treatment with the peptides on oxidative activity, free-radical processes, and the accumulation of lipid peroxidase byproducts in blood serum and brain samples collected from rats [3].

For the purpose of this study, 30 adult male Wistar rats weighing 230-250 grams were intraperitoneally injected with 2.5 ug/kg of epitalon and cortagen daily, for 5 days. Following treatment, blood plasma samples were collected from the animals that were then euthanized in order for brain tissue samples to be dissected and prepared. The parameters of antioxidant activity and free-radical oxidation were measured in the rat serum, as well as 10% brain homogenate in a phosphate buffer containing 105 mM KCL and 60 mM KH2PO4. The researchers then used conjugated dienes and Schiff’s bases to assess the intensity of the lipid peroxidase activity while the level of oxidative modification of proteins was assessed by observing the amino acid carbonyl derivative content in proteins following reaction with 2,4-dinitrophenylhydrazine [3].

Additionally, total antioxidant activity in the blood serum and brain tissues was evaluated through riboflavin chemiluminescence and protein content in the reaction mixture. The researchers also measured levels of antioxidant enzymes glutathione peroxidase and SOD through the inhibition of NBT reduction in a NADH-containing system in the presence of biological materials. This procedure was important to mention considering that the reduction of NBT is related to SOD inhibition and the release of superoxide radicals. Using NADH concentrations enzyme activity was measured using optical densities collected from samples obtained from experimental and control group animals [3].

 

Discussion

1) As it was previously mentioned, administration of cortagen resulted in a decrease in neuromas near the suture area in the mice, prompting the research team to measure action potential conduction velocity both at the suture site and in the regenerating fragment of the sciatic nerve. When the conduction velocity test was performed it was found that there was complete restoration in animals included in both the experimental group and the control group.

Action potential conductivity along the distal portion of the nerve was measured at 23.5 m/s in the control group animals and 22.8 m/s in the experimental group animals. Conductivity of the action potential measured through the suture was measured at 22.2 m/s in the control group animals and 24.0 m/s in the experimental group animals. The research team mentioned that the measurement process was facilitated by recording the action potential in vitro with standard registration conditions provided and a limited interelectrode distance on the platform the nerve was placed on. Based on these procedures the conductivity of the action potential was determined only along the most rapidly conducting nerve fibers that were then categorized into “group A” [1].

Single sensory fiber action potentials were analyzed by mechanically stimulating the skin of the bottom of the foot. The researchers looked at 50 different microfilaments that were separated from the tibial branch of the sciatic nerve. Among the 50 microfilaments assessed there were 16 nerve fibers in the experimental group animals and 12 nerve fibers in the control group animals that were able to provide adequate mechanical stimulation to the skin of the foot. These receptors were distributed across the surface of the sole and toe pads, suggesting that there was complete restoration of foot innervation by the regenerating sciatic nerve [1].

Additionally, in the rats treated with cortagen, the receptor sensitivity to mechanical stimuli to the sole of the foot was significantly increased in comparison to the control group of animals. However, while sensitivity was higher in the cortagen-treated animals, there were no significant differences in the distribution pattern of the receptors. That being said, the various dynamic and static components of trapezoid stimulus delivered to the skin of the foot revealed that there were different receptor type characteristics in the intact skin. The three main receptor characteristics identified were slowly adapting, rapidly adapting, and very rapidly adapting Pacinian-like receptors [1].

Figure 1: Changes in the perception threshold for the mechanical trapezoid stimulus applied to the skin of the rats’ foot.

Finally, when analyzing the action potential elicited by mechanical stimulation recorded from the single nerve fibers there was a decrease in the duration of the action potential. The research team noted that action potential duration from a single nerve fiber is variable and can be influenced by the functional properties of the fiber and the conductivity of the action potential along the single nerve fiber. This findings suggests that nerve impulse conduction along regenerating afferent nerves is facilitated by treatment of cortagen. Overall, the researchers concluded that the combination of these findings and those of their previous work on the efficacy of cortagen, assists in confirming the ability of the peptide to restore the function of injured nerve fibers that may have a diminished capacity for regeneration [1].

2) The research team of Tuchaninova et al examined the effects of cortagen on restoring functioning and facilitating regeneration in damaged sciatic nerve tissue. Functioning of the nerve was assessed by dissecting and suturing the sciatic nerve from male Wistar rats and placing the nerve onto a multielectrode platform in order to induce a measurable compound action potential. Based on the recorded data the researchers were able to calculate the conduction velocity of the sciatic nerve being tested. Overall, the results reported that stimulating the sciatic nerve induced an actional potential that travelled along the full nerve fragment [2].

In order to best reveal the effects of several different influencing factors on the restoration of nerve conductance in regenerating tissue, all electrical testing took place 4 weeks after dissection and suturing of the sciatic nerve occurred. This time period is considered optimal since the distance at which the sciatic nerve conduction was restored could be most accurately determined. That being said, the distance of nerve conduction restoration was shown to correspond to the length of the regeneration nerve fiber. However, the researchers mentioned that the value is not absolute considering that some regenerating microfiber are able to grow longer distances while their activity is shunted by the nerve trunk, not allowing them to contribute to an action potential [2].

The results of the compound action potential recording found that animals in the control group had significant variations between subjects in the growth rate of sciatic nerve fibers. These findings confirmed to the research team that the process of nerve regeneration with no other facilitatory factors is influenced by numerous factors including, sex, age, and degree or area of damage that may or may not be specific to the experiment. Hormonal and immunological factors that are not specified in the study are different from subject to subject and result in several individual variations throughout the neuronal regeneration process.

The animals included in the experimental group experienced limited variation in the growth rat of the sciatic nerve fibers. Treatment with cortagen was shown to enhance the regeneration process in animals with a low neuronal growth rate. Additionally, all of the regenerating nerve fibers in the animals treated with cortagen were shown to experience a significant increase in nerve length, as well as restored nerve conduction [2].

Latency and conduction velocity was calculated by the research team by using the compound action potential that was recorded at a distance of 12 mm from the reference electrode. At a distance of 12 mm, all rats demonstrated consistent compound action potentials. In comparison to intact rats, both animals in the experimental group and the control group had significantly lower conduction velocities, by 66% and 53%, respectively. Decreased conduction velocity is typical in the initial stages of the regeneration process due to small diameter and poor myelination of growing sciatic nerve fibers. However, the animals treated with cortagen experienced significantly higher conduction velocities in comparison to the vehicle-treated animals. These findings suggest that cortagen stimulates regeneration in damaged sciatic nerve fibers [2].

Overall, the findings collected from recorded data of compound action potentials revealed that treatment with cortagen facilitated regeneration and functional maturation of sciatic nerve fibers. This was represented by a 40% increase in conduction velocity, primarily in sensory and motor thick myelinated A-fibers. The research team concluded that the peptide elicits potent neurotrophic activity and promotes both functional and structural recovery of damaged afferent nerves [2].

3) After synthesizing the peptide derivatives cortagen and epitalon from the polypeptides cortexin and epithalamide, the research team of Kozina et al administered the peptides to male Wistar rats in order to examine changes in the development of lipid peroxidase byproducts. Treatment with both cortagen and epitalon was found to suppress the formation of lipid peroxidase byproducts. Epitalon elicited more potent effects, significantly decreasing both primary and final lipid peroxidase byproducts in the brain. Treatment with cortagen was also shown to drastically reduce levels of primarily lipid peroxidase byproducts, however, there was only a slight change noticed in the final byproducts. The reduction in byproduct formation was regulated primarily in the early stages of lipid peroxidase, resulting in a significant decrease in primary byproducts and a reduction trend of final byproduct development [3].

In addition to suppression of byproduct formation, the dynamics of the lipid peroxidase byproducts were observed in order to estimate the volume of the medium and the protein content. The collected data revealed that treatment with cortagen only had the potential to inhibit oxidative modification of proteins in the blood serum by approximately 15%. These findings shed light on the relationship between lipid peroxidase byproducts and oxidative modifications, revealing that the final byproducts form due to the presence of primary byproducts, as well as reactions between lipid peroxidase products and compounds containing free amino groups. This ultimately results in the formation of carbonyl groups causing oxidative modifications and oxidative stress that precedes lipid peroxidase [3].

Additional experimentation took place to further expand on the antioxidant effects of epitalon and cortagen. Both peptides were administered to the rats in doses ranging from 0.5 ug/ml to 5.0 ug/ml, and neither exhibited antioxidant effects in vitro. However, in vivo, there was clear evidence of antioxidant activity in blood serum and brain tissue. Treatment with cortagen resulted in a decrease in total antioxidant activity primarily in blood serum samples, as well as inhibition of antioxidant enzymes, glutathione peroxidase in the brain and SOD in blood serum. The research team thought it was important to note that cortagen and epitalon elicited similar effects on several free-radical oxidation parameters due to their structural similarity.

Overall, the researchers were able to conclude that the benefits elicited by the peptides epitalon and cortagen are due to the development of lipid peroxidase byproducts and the resulting oxidative modification of proteins and compensatory diminishment of total antioxidant activity. Administration of the peptides also resulted in reduced development of free oxygen radicals causing oxidative destruction of lipids and proteins [3].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Kolosova LI, Moiseeva AB, Turchaninova LN, et al. The delayed effect of cortagen on the restoration of injured nerve function. Dokl Biol Sci. 2002;384:183-184. doi:10.1023/a:1016098302564

[2] Turchaninova LN, Kolosova LI, Malinin VV, Moiseeva AB, Nozdrachev AD, Khavinson VK. Effect of tetrapeptide cortagen on regeneration of sciatic nerve. Bull Exp Biol Med. 2000;130(12):1172-1174.

[3] Kozina LS. Effects of bioactive tetrapeptides on free-radical processes. Bull Exp Biol Med. 2007 Jun;143(6):744-6. English, Russian. doi: 10.1007/s10517-007-0230-8. PMID: 18239817.

 

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