RU2538621C2 - Method for stimulating restoration of tissue innervation following injuries and ischemia by means of vector construct - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: therapeutically effective amount of a recombinant plasmid with a nucleotide sequence of SEQ ID NO:1, which contains a modified nucleotide sequence coding human BDNF, and the Kozak consensus sequence, is introduced into an involved muscle; pVax1 Invitrogen is used as a plasmid vector.

EFFECT: invention discloses an effective method to accelerate the restoration of the structure and conductivity of peripheral nerves after an injury, by transfection of the muscles innervated by the injured nerve, by means of a new plasmid construct coding the brain-derived neurotrophic factor.

3 cl, 5 dwg, 4 ex

Description

The present invention relates to the field of genetic engineering and gene therapy and can be used in regenerative medicine, traumatology, transplantology and neurobiology to stimulate the growth and regeneration of nerves and restore the innervation of ischemic tissues. A method is proposed that allows to accelerate the restoration of the structure and conductivity of peripheral nerves after injuries by transfecting the muscles innervated by the damaged nerve with a new plasmid construct encoding brain neurotrophic factor (BDNF).

Introduction

Disorders of peripheral innervation caused by trauma, ischemia and neurodegenerative diseases are the most important cause of temporary and permanent disability of the Russian population of working age [1]. Inadequate restoration of innervation also significantly reduces the successful engraftment of transplanted tissues and organs (liver, kidneys, heart).

Stimulation of the growth and restoration of the peripheral nerve is the result of a complex process, including many physiological and biochemical reactions. For its implementation, first of all, it is necessary to support the survival and regeneration of neurons and to regulate the direction of axon growth, in which neurotrophic factors play a major role: cerebral neurotrophic factor (BDNF), glial growth factor (GDNF) and nerve growth factor (NGF).

Thus, based on a theoretical analysis of the issue, we can conclude that a local increase in the production of neurotrophic factors can have a stimulating effect on the process of nerve regeneration and restoration of peripheral innervation in the lesion site.

State of the art

One of the most developed approaches to solving the issue of local provision of certain organs, tissues and their individual zones with the necessary polypeptide products (hormones, enzymes, growth factors, etc.) is the use of vector constructs that include genes that encode the desired proteins, in this case key factors stimulating nerve growth and restoration of peripheral innervation.

Known methods of stimulating the restoration of peripheral innervation, involving the introduction to the patient of a sequence encoding BDNF, as part of adeno- and adeno-associated viruses, as well as lentiviral constructs [2]. Earlier in our laboratory, a method was developed to stimulate the restoration of peripheral innervation by introducing a non-viral vector construct containing an optimized human BDNF cDNA sequence.

Due to the above reasons, the work of Alrashdan [3] and our own data [4, 5], which describes methods for the regeneration of a peripheral nerve after an injury, can be considered analogues of the present invention.

The method proposed by Alrashdan et al. Is based on stimulation of the process by injection into the area of the innervation disorder of the adenovirus construct containing the BDNF gene (BDNF-Ad), with the aim of locally increasing the content of this cell factor involved in the restoration of nerve cells. Moreover, in addition to the fact that the use of recombinant viruses for transfection cannot be considered absolutely safe, the proposed use of the BDNF-Ad construct was also not effective enough: the recovery period of innervation, according to the authors, was approximately 3 weeks.

Regarding the method proposed earlier, it should be noted that despite the fact that it was possible to demonstrate the positive effect of the developed non-viral construct on the restoration of peripheral innervation, the effect was not sufficiently pronounced.

In this regard, an attempt was made to improve the method of stimulating the restoration of peripheral innervation using the method of recombinant DNA in the direction of increasing its efficiency.

Disclosure of invention

In developing the present invention, the following facts have been taken into account.

The first one. Successful stimulation of growth and recovery of the peripheral nerve requires support for the survival and regeneration of neurons, as well as regulation of the direction of axon growth. This function is performed by neurotrophic factors such as BDNF, glial growth factor (GDNF) and nerve growth factor (NGF). Accordingly, we can conclude that stimulation of the growth of nerve endings and restoration of nerves with a high degree of probability can be achieved by providing long-term production of neurotrophic factors in the lesion.

The second one. It is known that long-term production of neurotrophic factors can be achieved by introducing into the damaged tissue genetic engineering constructs containing cDNA of these proteins. The human BDNF cDNA sequence is characterized by a high content of so-called “Rare triplets”, which may negatively affect the efficiency of its expression. Earlier in our laboratory, such triplets were replaced by those that are most often found in human genes, without changing the encoded amino acid sequence, which ensured long-term production of this protein in damaged skeletal muscles [4].

A further increase in the efficiency of transgene production can be achieved by introducing a Kozak consensus sequence into its structure [6].

Given the above facts, achieve increased efficiency expression of BDNF in the lesion sites can be achieved by introducing a Kozak consensus sequence into the expression vector.

The third. To successfully stimulate the restoration of innervation in ischemic tissue, it is necessary to ensure a high level of neurotrophic factor production. Accordingly, the use of a plasmid construct encoding the brain neurotrophic factor and providing the maximum possible transgene production could be a high probability of stimulating the restoration of innervation in ischemic tissues.

With this in mind, the solution of the task involved:

a) creating a plasmid construct containing optimized brain neurotrophic factor cDNA (BDNF), into which the Kozak consensus sequence has been introduced;

b) evaluation of the expression efficiency of the created plasmid construct in vitro; and

c) determining the result of the introduction of the plasmid construct on the effectiveness of the recovery of the peripheral nerve after injury, as well as on the restoration of innervation in ischemic tissue.

When carrying out the invention, a recombinant plasmid with SEQ ID NO: 1, shown in the sequence listing, containing the optimized BDNF cDNA sequence (highlighted in gray shading and bold on SEQ ID NO: 1) and the Kozak consensus sequence (double highlighted) was first constructed (Example 1) underlined on SEQ ID NO: 1). In this case, the best results were obtained when pVax1 (# V260-20, Invitrogen) was used as the plasmid vector, however, other plasmid vectors, which are characterized by high copy replication in E. coli and a high level of expression of the cloned gene in cells, can be used for cloning mammals.

As a result of the study of the expression of the obtained plasmid constructs, it was shown that 48 hours after transfection of cells, secretion of the corresponding proteins into the culture medium is observed. Due to the inclusion of optimized nucleotide sequences, the use of plasmid constructs for the expression of human BDNF allowed us to achieve an increase in the production of target proteins by about 3 times (example 2).

On the model of traumatic damage to the peripheral nerve of the mouse, experimental confirmation was obtained that the constructed plasmid construct, after introduction into the muscle innervated by the damaged nerve, significantly accelerates the restoration of the structure and conductivity of the damaged nerve. It was found that the introduction of the plasmid pVax1-K-BDNF provides an additional 1.5-2 fold (in comparison with the use of a plasmid that does not contain the Kozak consensus sequence, pVax1-BDNF) acceleration of the restoration of the structure and conductivity of the nerve, and hence the innervation corresponding skeletal muscles (example 3).

For the first time, the created plasmid construct was used on the mouse hind limb ischemia model. The introduction of the plasmid construct pVax1-K-BDNF allowed us to achieve an increase in the number and total length of axons in the ischemic muscle by 2-3 times, in contrast to the previously developed plasmid pVax1-BDNF (example 4).

Thus, as a result of the creation of the present invention, a method for stimulating recovery of muscle innervation after trauma or ischemia is developed, the essence of which is that a therapeutically effective amount of a plasmid vector containing the optimized nucleotide sequence of SEQ ID NO: 1 is introduced into a muscle innervated by a damaged nerve. encoding BDNF.

In this case, the main technical result is to increase the efficiency of the method: when using essentially the same model of trauma to the peripheral nerve, the values of the main indicators of tissue innervation increased approximately twofold in comparison with the prototype. Additional results of this method should be considered the possibility of using the developed design to stimulate the innervation of ischemic tissues and the rejection of the use of unsafe adenoviral structures.

Brief Description of the Drawings

Figure 1. Analysis of the production efficiency of brain neurotrophic factor by cells transfected with plasmid constructs.

Figure 2. Restoration of the function of the extensor muscles of the foot. On the y-axis - the value of PFI, conventional units; abscissa - time after damage, days.

Figure 3. Restoring the number of axons in a damaged nerve. A. - the nerve of the mouse, which was introduced plasmid that does not contain a transgene. B. - the nerve of the mouse, which was injected with the plasmid construct pVax1-BDNF. B. - the nerve of the mouse, which was injected with the plasmid construct pVax1-K-BDNF. The number of axons was evaluated by staining with rabbit antibodies to the axon cytoskeleton, protein NF200. G. - the results of a morphometric analysis of the number of axons on a cut of a damaged nerve. The ordinate axis is the number of axons, pcs.

Figure 4. Restoring nerve conduction. A. - restoration of the latent period of SPDN, along the ordinate axis - the duration of the latent period, ms. B. - restoration of the SPDN amplitude, along the ordinate axis - the SPDN amplitude, mV.

Figure 5. Restoring the number of nerve fibers in ischemic tissue. A. - ischemic muscle of the mouse, which was introduced plasmid that does not contain a transgene. B. - ischemic muscle of the mouse, which was injected with the plasmid construct pVax1-BDNF. B. - ischemic muscle of a mouse injected with the plasmid construct pVax1-K-BDNF. The number of nerve fibers was evaluated by staining with rabbit antibodies to the axon cytoskeleton, protein NF200. G. - restoration of the number of nerve fibers on transverse sections of the ischemic muscle, along the ordinate axis - the number of nerve fibers, pcs .; abscissa - time after damage, days. D. - restoration of the total length of nerve fibers in longitudinal sections of the ischemic muscle, along the ordinate axis - the total length of nerve, μm; abscissa - time after damage, days.

The implementation of the invention

In the implementation of the invention, in addition to the methods described in detail in the following examples, standard technologies of genetic engineering, culturing mammalian cells and the previously described methods for analyzing nerve regeneration after injury were used.

Example 1. Creating a plasmid construct encoding a human BDNF

As previously described, an optimized plasmid construct encoding human BDNF cDNA was designed as follows. Using the codon frequencies found on the website http://del.mediaglyphs.org/mg/bf/nucs_aa.html, among the triplets of nucleotides encoding the amino acids of the protein sequence of the natural BDNF cDNA, those that are found most rarely in human genes . Identified rare triplets, as well as neighboring triplets, were exchanged for triplets encoding the same amino acids, but which are most often found in human genes.

In order to increase the translation efficiency of BDNF mRNA, the Kozak consensus sequence was inserted into cDNA [6]. Namely, 2 triplets were introduced, up to the start codon 3'-GCCACC-5 '. To obtain a “strong” Kozak sequence, it was also necessary to introduce G immediately after the start codon. For this, a GTG triplet encoding the amino acid valine was introduced into the sequence. This approach made it possible to introduce the “strong” Kozak sequence into the BDNF cDNA, while avoiding the shift of the reading frame. The obtained nucleotide sequence encoding BDNF is presented in figure 1. This sequence was obtained using direct chemical synthesis of the fragments and their subsequent assembly. The identity of the expected sequence was determined by sequencing.

To clone the optimized cDNA into the expression vector, sequences recognized by the EcoRI and EcoRV endonucleases (5`-GAATTC-3` and 5`-GATATC-3`, respectively) were added to its expression vector at its 5` and 3` ends. In this example, plasmid pVax1 (# V260-20, Invitrogen) was used as a vector, however, other plasmid vectors, which are characterized by high copy replication in E. coli and a high level of expression of the cloned gene in mammalian cells, could be used for cloning.

Example 2

Analysis of the effectiveness of the obtained plasmid construct

The expression efficiency of the developed plasmid was determined by the production of the target protein by HEK293 cells transfected with this plasmid. HEK293 cells seeded in wells of a 24-well plate (50,000 cells per well) in DMEM medium containing 2% fetal calf serum were transfected with plasmids pVax1, pVax1-BDNF or pVax1-K-BDNF. Transfection was performed using 1 μg of one of the plasmid DNAs and 1 μl of reagent Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer's protocol. 24 hours after transfection, the culture medium was replaced with fresh. The concentration of BDNF in the medium was analyzed after the next 48 hours of cultivation.

The concentration of BDNF in the culture medium was measured using a ChemikineTM BDNF ELISA Kit (Millipore, USA) in accordance with the manufacturer's protocol. According to these measurements, the production of BDNF into the culture medium of cells transfected with a Kozak-free plasmid is not more than 2.5 ng / ml. Using a plasmid containing optimized cDNA and a Kozak sequence, the concentration of BDNF in the culture medium was about 5 ng / ml. When using a plasmid with optimized cDNA and a Kozak sequence, the yield of the target protein is approximately 2 times higher than using the previously developed plasmid, which indicates the high efficiency of the plasmid pVax1-K-BDNF, which includes the Kozak consensus sequence (Fig. 2).

Example 3

The effect of plasmid pVax1-K-BDNF on the restoration of the peripheral nerve and innervation of the muscle after injury

To study the process of regeneration of the peripheral nerve, a model of damage to the common fibular nerve was used, as described previously [4, 5]. Immediately after nerve damage, 60 to 200 μg of plasmid DNA pVax1, pVax1-BDNF or pVax1-K-BDNF was introduced into the anterior tibial muscle. This therapeutically effective concentration was selected according to the results of preliminary experiments.

Recovery of muscle innervation was evaluated after 1, 2, 4, and 7 days by restoring the function of the extensor muscles of the toes of the toes. Restoration of the function of the extensor muscles of the foot was analyzed by measuring the functional index of the peroneal nerve (PFI) E, as described previously [5]. It was found that after 2 days in animals that were injected with pVax1-K-BDNF, the PFI value was about 1.6 times higher than in the control, and 1.4 times higher than in animals that were injected plasmid pVax1-BDNF (figure 3).

The restoration of the structural integrity of the nerve was evaluated using immunofluorescence staining of frozen sections of damaged nerves 4 days after the damage as described previously [4, 5]. Approximately 2.5 times more axons containing NF200 were observed in animals injected with pVax1-K-BDNF already 4 days after injury compared with nerves of animals that were injected with plasmid pVax1 without transgene (p <0.05 ; n = 18) and about 1.3 times more compared with animals that were injected with the plasmid pVax1-BDNF. These data indicate faster growth and better axon survival in damaged nerves of animals injected with pVax1-K-BDNF.

Recovery of function of sensitive nerve fibers was evaluated using evoked action potentials recorded from the surface branch of the common peroneal nerve, as described previously [4, 5]. The data obtained were analyzed as follows. The registered SPDN contains two parameters that make it possible to judge the degree of nerve recovery: latent period and amplitude.

The latent period is the time interval from the moment of nerve stimulation to the moment of registration of the evoked action potential, measured in milliseconds (ms). Based on the definition, it is clear that the latent period characterizes the rate of stimulation of the nerve, and the magnitude of the latent period is inversely proportional to the speed of the excitation of the nerve. Thus, the shortening of the latent period indicates a higher rate of evoked potentials, which, in turn, indicates the degree of myelination for myelinated nerve fibers or an increase in the thickness of non-myelinated nerve fibers.

The amplitude characterizes the number of nerve fibers involved in conducting the excitation; measured in millivolts (mV). The larger the amplitude, the greater the number of restored nerve fibers.

By recording evoked nerve action potentials 7 days after the damage, it was possible to show that injection of the pVax1-K-BDNF genetic construct stimulates nerve repair. Injection of the plasmid caused a decrease in the latent period of about 1.6 times and an increase in the SPDN amplitude by about 3 times compared with the corresponding values of the SPDN of mice that received plasmid pVax1 without transgene (Fig. 5).

Moreover, injection of plasmid pVax1-K-BDNF led to a more pronounced restoration of nerve conduction compared to the previously developed plasmid pVax1-BDNF. So, in animals treated with pVax1-K-BDNF, the latent period was shortened by about 1.5 times, and the increase in the SPDN amplitude was about 1.3 times larger than the corresponding values for mice injected with pVax1-BDNF. These data indicate that injection of pVax1-K-BDNF stimulates the restoration of nerve fiber conduction.

Example 4

The effect of plasmid pVax1-K-BDNF on the restoration of muscle innervation after ischemia

To study the process of restoration of innervation in ischemic muscles of the hind limb, there are a number of methods based on clamping or excision of the femoral artery of the mouse. In this case, they used their own modification of the known models of hind limb ischemia in rodents (Takeshita, 1994; Tsurumi, 1996). Male C57H1 mice weighing 30 g were used in the work. All experimental animal procedures were in accordance with the Guidelines for the Control of Animal Experiments, approved by the local ethics committee. The operation was performed under anesthesia by inhalation of 2.5% avertin. A longitudinal incision was made of the skin of the left thigh of the mouse from the inguinal ligament to the knee region. The artery was isolated completely from the onset of bifurcation of the abdominal aorta to the division into a.poplitae and a.saphenous and its branches, including a.epigastrica inferior, a.femoralis profunda, a.circumflexus lateralis, ligated and cut off. A. poplitae and a.saphenous were also bandaged and cut off. The femoral artery was removed all the way from the inguinal ligament to the bifurcation on a.poplitae and a.saphenous.

The experiments showed that this method of damage is optimal for creating ischemia of the hind limb of the mouse and can achieve a decrease in blood flow to less than 10% of the control. Other methods of inducing ischemia of the hind limb, for example, by ligation of the femoral artery of the mouse, turned out to be less effective, since they led only to a short-term disturbance of blood flow in the limb and a quick spontaneous restoration of its functional activity, which would not allow to evaluate the effect of the introduction of the gene drug.

Studies of the therapeutic effectiveness of Innervine to restore the innervation of the ischemic muscle were performed for 11 days after the induction of hind limb ischemia. For this, solutions of a plasmid construct containing BDNF cDNA or a control plasmid that does not carry neurotrophic factor cDNA were injected in sterile phosphate-buffered saline the day after surgery by percutaneous injection into limb muscles - 1 injection of 60 μl, total 200 μg of DIC per mouse . After 4, 7, and 11 days, m.tibialis anterior was isolated from lethally anesthetized animals and frozen in Tissue-Tek (Sakura Finetek) freezing medium by smooth immersion in liquid nitrogen. Transverse and longitudinal cryosections 6 μm thick were prepared from frozen muscles.

The criteria for assessing the effectiveness of Innervin for restoring the innervation of damaged muscles are the number and total length of nerve fibers, estimated using immunofluorescence staining of cryosections of muscles using specific antibodies to the cytoskeleton protein of mature axons NF200. Cryosections were stained as follows: sections after drying on a glass slide were fixed with 4% formalin on phosphate buffer for 5 minutes, then washed from the fixative, placed in a 0.05 M solution of ammonium acetate on phosphate buffer to block autofluorescence of the sections for 10 minutes and in 1% bovine serum albumin solution in phosphate buffer to block non-specific antibody binding. Primary and secondary antibodies were diluted in phosphate-buffered saline containing 0.1% Triton X-100 detergent and 5% normal goat serum. Primary rabbit antibodies to the mature axon cytoskeleton protein NF200 (Abcam, dilution 1:80) were applied to the sections. Some sections were stained with rabbit non-specific IgG immunoglobulins as a negative control (Thermo scientific). Sections coated with primary antibodies were incubated in a humid chamber at 4 ° C overnight. Next, the sections were washed in phosphate-buffered saline (pH 7.0) 3 times for 5 min and secondary antibodies were applied onto them: goat antibodies against rabbit immunoglobulins conjugated with AlexaFluor 488 fluorochrome (Invitrogen). Sections were incubated in a dark place in a humid chamber for 1 hour at room temperature. Then the sections, protected from light, were washed in phosphate-buffered saline (pH 7.0). Next, the sections, protected from light, were incubated for 2 minutes, and then again washed in phosphate-buffered saline (pH 7.0) 3 times for 5 minutes. Excess fluid was shaken off from the sections and enclosed in a special, non-fluorescent Mounting Media (Vector).

The obtained sections were recorded using a Leica6000 fluorescence microscope equipped with a CCD camera (Leica Microsystems). The excitation of AlexaFluor 488 dye was carried out at a wavelength of 488 nm, and the registration of induced excitation was carried out at a wavelength of 546 nm. The number of stained axons and their length in the obtained images was evaluated using the Metamorph 7.1 program.

When analyzing cross-sections of ischemic muscles stained with antibodies against the cytoskeleton protein of mature axons NF200, it was found that the number of nerve fibers in the muscles of animals that were injected with the gene drug Innerwin was higher compared to mice that received the control plasmid 7 days after induction ischemia and introduction of plasmids, however, the detected difference was not statistically significant. 11 days after the introduction of plasmids, the number of nerve fibers in the muscles of animals that were injected with the gene drug Innerwin was approximately 4 times higher compared with mice that received the control plasmid (Fig.6).

When analyzing longitudinal sections of muscles stained with antibodies against the cytoskeleton protein of mature axons NF200, it was found that the total length of nerve fibers in the muscles of animals that were injected with the Innervin gene drug was longer already 4 days after the induction of ischemia and the introduction of plasmids. Differences persisted after 7 days of observation, and after 11 days, the total length of nerve fibers in animals that were injected with the Innerwin gene drug was approximately 3 times greater than in mice that received the control plasmid.

Conclusion

As a result of the studies, it was first shown that the injection of the plasmid construct pVax1-K-BDNF into the muscle innervated by the damaged nerve, followed by electroporation, stimulates the regeneration of:

approximately 1.6 times in comparison with the control and 1.4 times in comparison with the previously developed plasmid, the restoration of innervation is accelerated (compare the curves in Fig. 3), which is accompanied by an increase in the number of axons in the damaged nerve by 2.5 and 1.3 times, respectively (compare the columns in FIG. 4), a decrease in the latent period of about 1.6 and 1.5 times, respectively, as well as an increase of almost three and 1.3 times, respectively, of the amplitude of the total potential action of the nerve (compare the columns in FIG. .5).

It was found that the introduction of the plasmid pVax1-K-BDNF into the ischemic muscle with subsequent electroporation stimulates the restoration of innervation, which is manifested in an increase in the number of axons by 7 times and the total length of nerve fibers by more than 3 times (compare the curves in Fig.6).

Based on the obtained data, it was concluded that the stimulation of restoration of the nerve structure and function after injury, as well as the innervation of ischemic tissue, can be achieved by introducing a plasmid construct containing the optimized human BDNF nucleotide sequence and Kozak consensus sequence. The high production of the target protein achieved as a result of the modification provided not only the possibility of using this plasmid to stimulate the restoration of nerves after an injury, but also allowed to stimulate the restoration of the innervation of the ischemic muscle.

Bibliography

1. Odinak M.M., Zhivolupov S.A. Diseases and injuries of the peripheral nervous system. St. Petersburg, SpetsLit., 2009.

2. Kwon BK, Liu J, Lam C, Plunet W, Oschipok LW, Hauswirth W, Di Polo A, Blesch A, Tetzlaff W. Brain-derived neurotrophic factor gene transfer with adeno-associated viral and lentiviral vectors prevents rubrospinal neuronal atrophy and stimulates regeneration-associated gene expression after acute cervical spinal cord injury. Spine (Phila Pa 1976). 2007; 32 (11): 1164-73.

3. Alrashdan MS, Sung MA, Kim Kwon Y, Chung HJ, Kim SJ, Lee JH. Effects of combining electrical stimulation with BDNF gene transfer on the regeneration of crushed rat sciatic nerve. Acta Neurochir (Wien), 2011 (on line 06/09/2011)

4. Patent application "Method of stimulating the restoration of peripheral tissue innervation using vector designs", Application 2011143050, Submission date October 25, 2011, Applicant: State Educational and Scientific Institution Faculty of Fundamental Medicine, Moscow State University named after M.V. Lomonosov

5. Lopatina T, Kalinina N, Karagyaur M, Stambolsky D, Rubina K, Revischin A, Pavlova G, Parfyonova Y, Tkachuk V. Adipose-derived stem cells stimulate regeneration of peripheral nerves: BDNF secreted by these cells promotes nerve healing and axon growth de novo. Plos one. 2011; 6 (3): e17899.

6. Kozak M (October 1987). "An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs." Nucleic Acids Res. 15 (20): 8125-8148.

Claims (3)

1. A method of stimulating the restoration of the innervation of damaged tissue by introducing into the damage region a vector construct that provides a high level of expression of a factor involved in the nerve regeneration process, which is characterized in that a therapeutically effective amount of a recombinant plasmid with the nucleotide sequence of SEQ ID NO is introduced into the damaged muscle: 1, which contains a modified nucleotide sequence encoding a human BDNF, and a Kozak sequence, as a plasmid vector pVax1 Invitrogen is used. 2. The method according to claim 1, in which the damage is a trauma to the peripheral nerve. 3. The method according to claim 1, in which the damage is ischemic damage to the limb.

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