Keywords nerve regeneration - peripheral nerve - immunosuppressive agents - FK506 - tacrolimus
- cyclosporine
Introduction
Peripheral nerves consist of structures that can be affected by injuries that can
result in important motor and sensory impairments.[1 ] Peripheral nerve injuries (PNIs) cause interruption of the continuity of structures
and block the propagation of nerve impulses, consequently affecting the functional
capacity of individuals.[2 ]
[3 ]
[4 ]
[5 ]
[6 ]
Peripheral nerve injuries are more frequent in the upper limbs, causing problems mainly
in the functional abilities of the hand. Regeneration of the lesion in the post-trauma
is of great complexity and the individual can present severe sequelae. Thus, PNIs,
in addition to promoting functional impairments in the quality of life of the subjects,
directly affect their finances due to the impossibility of carrying out daily activities,
and indirectly due to the supporting care related to the treatment.[7 ]
Therapeutic strategies for the treatment of PNIs represent a major challenge in regenerative
medicine due to the complex biological conditions and to the lack of biomaterials
for an effective repair of the nerves.[8 ] The treatment is classified into two modalities and depends exclusively on the type
of lesion presented: in more severe situations, such as neurotmesis, surgical treatments
are recommended, whereas for less severe conditions, such as axonotmesis and neuropraxia,
pharmacological conservative treatments are indicated.[5 ]
The development of drugs that promote the rate of nerve regeneration and increase
the degree of functional restitution after injury can bring extremely important benefits
for patients with PNI. Of these drugs, immunosuppressants were initially used to prevent
allograft rejection.[9 ]
[10 ]
Tacrolimus, also known as FK506 or Fujimycin (C44 H69 O12 ), is a drug from the macrolide group, which was approved by the FDA (Food and Drug
Administration) for the prevention of allograft rejection, but it also has nerve regeneration
properties via immunosuppression.[11 ]
Another important immunosuppressant to prevent organ transplant rejections is cyclosporine,
which can significantly reduce morbidity when compared with early immunosuppression
methods. Regarding nerve regeneration, the mechanism of action of cyclosporine is
still contradictory. Some researchers reveal that the local effect of cyclosporin
A (CsA) on peripheral nerve regeneration has been investigated after peripheral nerve
transection in experimental models immunosuppressed with CsA. Most of these studies
focused on the local effect of CsA on peripheral nerve regeneration rather than on
allograft survival.[12 ]
[13 ]
[14 ]
[15 ]
Considering the promising action of immunosuppressants, there is no consensus in the
literature about the effectiveness of these drugs. Therefore, the objective of the
present investigation is to review the scientific evidence of the repercussion of
the use of immunosuppressants in the regeneration of PNIs.
Materials and Methods
The present work was conducted according to the PRISMA guidelines for reporting systematic
reviews and was registered on the PROSPERO platform, which is available on the website
(http://www.crd.york.ac.uk/PROSPERO ).
The search strategy was performed by identifying the studies, without language restriction,
published from the inception to September 2018 in the following electronic databases:
MEDLINE (via PubMed), EMBASE, Cochrane Central Register of Controlled Trials, ISI
Web of Science (http://www.webofknowledge.com ), Oxford Pain Relief Database, and LILACS. As grey literature, potentially eligible
articles were also searched in the bibliographic references of the materials searched
in the aforementioned databases. First, the search was performed in MEDLINE and the
following searches were adapted to the other databases.
The search terms used were: “Peripheral Nerve Injuries ” (mesh), “Peripheral Nerve Injury ,” “Nerve Injuries, Peripheral ,” “Nerve Injury, Peripheral .” For intervention: “tacrolimus ” (mesh), “Prograf ,” “Prograft ,” “FR900506 ,” “FR
900506 ,” “FR900506 ,” “Anhydrous
tacrolimus ,” “tacrolimus , Anhydrous ,” “tacrolimus
Anhydrous ,” “Anhydrous , tacrolimus ,” “FK -506 ,” “FK
506 ,” “FK506 ,” “Cyclosporine ,” “Cyclosporin ,” “Cyclosporine
A ,” “Ciclosporin ,” “Cyclosporin
A ,” “Neoral ,” “Sandimmun
Neoral ,” “CyA -NOF ,” “CyA
NOF ,” “Sandimmune ,” “Sandimmun ,” “CsA -Neoral ,” “CsA
Neoral ,” “CsANeoral ,” “OL
27-400 ,” “OL
27
400 ,” “OL
27400 ,” “Cyclosporins ,” “Immunosuppressive
Agents ,” “Agents , Immunosuppressive ,” “Immunosuppressants .” For outcomes: “Nerve
Regeneration (mesh),” “Nerve
Regenerations ,” “Regeneration , Nerve ,” “Regenerations , Nerve ,” “Neuronal
Protection ,” “Protection , Neuronal ,” “Neural
Protection ,” “Protection , Neural ,” “Neuron
Protection ,” “Protection , Neuron ”.
The PICO strategy used in the present review was: P, peripheral nerve injury; I, the
use of immunosuppressants tacrolimus (FK-506) and/or CsA; C, the use of treatment
without immunosuppressants; and O, outcomes related to nerve regeneration. The present
review included all types of study designs in which the intervention is described
as using the immunosuppressants tacrolimus (FK-506) and/or CsA, and in which the outcomes
evaluated are related to peripheral nerve regeneration/neuroprotection. Reviews, duplicate
studies, and editorial studies were excluded, in addition to in vitro investigations,
studies performed in the central nervous system, and studies of degenerative nerve
diseases. In cases of duplicate studies, the one with the largest sample size was
considered.
Any type of outcome that indicates or measures nerve regeneration and/or neuroprotection
after peripheral nerve injury was accepted; histological morphometric and immunohistochemical
parameters of nerve regeneration, whether conventional or computed, such as counting
motor neurons (myelinated axons) by microscopy, measuring myelin sheath thickness
(morphometric analysis of axonal caliber), calculating the axon regeneration rate
determined by radiolabelling, body weight and muscle mass gain, electroneurophysiological
outcomes, and functional outcomes by assessment of motricity and sensitivity measured
by usual or unusual tests.
The selection of studies and data extraction took place as follows: two authors worked
independently and verified the abstract and title of the search results. Differences
were resolved by discussion or consultation with a third author for consensus. All
potentially relevant articles were investigated as full text. When there was no agreement,
the third author who did not initially review the articles made the final decision.
For studies that met the inclusion criteria, two authors independently extracted data
using standardized data extraction models.
The instructions from the Cochrane Handbook for Systematic Reviews of Interventions
were followed to assess the risk of bias.
Two authors independently handled the risk of bias in the included investigations
by considering the following items: generation of proper sequence (selection bias);
allocation sequence adequately concealed (selection bias); whether knowledge of assigned
interventions was correctly warned during the study; evaluation of participants and
staff (performance bias) evaluators of outcomes (detection bias); whether incomplete
outcome data were adequately addressed (attrition bias); whether the study reports
were free from the suggestion of selective reporting of results (reporting bias).
The present study was apparently free of other problems that could put it at risk
for bias. Disagreements arising from different interpretations were resolved by consensus.
When additional information was needed for the review, the authors reassessed the
study as soon as this information was made available by the authors of the investigated
article.
Statistical analysis was performed using R 3.6.0 statistical software and RStudio
interface version 1.2.1335 (R Foundation, Vienna, Austria). The meta-analysis was
performed using the "meta" package and the "metacont." The random-effects inverse-variance
method was used to aggregate the results. Studies that had multiple intervention and/or
control groups had mean and standard deviation results grouped according to the methodology
described by Higgins et al..[16 ]
Hedges g was used to estimate the effect size representing standardized mean differences
(SMDs). Heterogeneity between studies was assessed using the Q test and the I2 test. The I2 test represents the amount of total variation that is explained by the variation
between studies. I2 values of ∼ 25, 50, and 75% indicate low, moderate, and high heterogeneity, respectively.
All meta-analyses performed showed heterogeneity in the studies; therefore, the effect
size presented in the random-effects model indicator should be used.
Results
After identification of the articles, removal of duplicates, and exclusion following
the eligibility criteria, 56 studies were included in the present research.
Furthermore, most authors chose to injure the sciatic[15 ]
[17 ]
[18 ] or tibial[19 ]
[20 ]
[21 ]
[22 ]
[23 ]
[24 ]
[25 ] nerve and the time of the studies ranged from 7 days[26 ] to 1 year of observation.[27 ]
Even though, for most authors, the administration of tacrolimus or cyclosporine does
not present side effects, some articles report episodes of rejection and adverse effects.[10 ]
[19 ]
[28 ]
Of all studies, the most recurrent type of anaesthetic is pentobarbital[4 ]
[10 ]
[17 ]
[21 ]
[26 ]
[29 ]
[30 ]
[31 ]
[32 ]
[33 ] or ketamine[15 ]
[19 ]
[20 ]
[24 ]
[25 ]
[27 ]
[34 ]
[35 ]
[36 ]
[37 ]
[38 ] associated with medetomidine[20 ]
[25 ]
[35 ] or xylazine.[15 ]
[18 ]
[19 ]
[27 ]
[36 ]
[37 ]
[38 ]
Of the 56 articles in question, only 22 were selected for meta-analysis ([Supplementary material ] – available online). The results obtained through the analysis of the studies are
shown in the following figures.
As shown in [Figure 1 ], the combined SMD in the included studies was 1.66 (95% confidence interval [CI]:
0.93–2.39; p < 0.01), suggesting the protective effect of the use of tacrolimus regarding regeneration
of the number of myelinated axons. The I2 value was 89%, indicating high heterogeneity.
Fig. 1 Rate of tacrolimus use with the number of myelinated axons.
[Figure 2 ] shows an SMD of 1.70 (95%CI: 0.78–2.62; p < 0.01), indicating that a tacrolimus dose ≤ 2 mg exhibited a significant effect in
the number of myelinated axons.
Fig. 2 Rate of tacrolimus use (dose ≤ 2 mg) with the number of myelinated axons.
[Figure 3 ] shows that tacrolimus at a dose > 2 mg also exhibited a significant relation with
the number of myelinated axons, as the SMD was 1.54 (95%CI: 0.06–3.03; p < 0.01).
Fig. 3 Rate of tacrolimus use (dose > 2 mg) with the number of myelinated axons.
When the use of cyclosporine for nerve regeneration was evaluated, the SMD value was
0.40 (95%CI: - 0.38–1.18; p = 0.08), showing no significant result of the use of this immunosuppressant in terms
of number of myelinated axons in general. When specifically analysing the use of tacrolimus
and myelin thickness, the SMD was 3.85 (95%CI = 2.00–5.71; p < 0.01), suggesting the existence of a significant relationship between both variables.
Also, it was found that the use of tacrolimus was significantly correlated with muscle
weight, since the SMD was 2.45 (95%CI = 0.68–4.22; p < 0.01).
It is suggested that there is a significant benefit for the sciatic functional index
using tacrolimus, since an SMD of 3.34 was obtained (95%CI = 1.44–5.24; p < 0.01). When we evaluate the use of tacrolimus at a dose ≤ 2 mg and the sciatic
functional index, an SMD of 3.76 was obtained (95%CI = 1.11–6.41; p < 0.01), suggesting a significant relationship. When using a tacrolimus dose of 2 mg,
the SMD was 2.74 (95%CI = - 0.59–6.07; p < 0.01), indicating that there is a significant association between the use of tacrolimus
at this dose and the sciatic functional index.
Discussion
The present systematic review evidences that the use of immunosuppressants in the
regeneration of PNIs promotes an increase in the number of myelinated axons in general,
regardless of the administered dose. In addition, it ensures greater myelin thickness,
muscle weight and restoration of the sciatic functional index. However, heterogeneity
was high in most analyses performed.
In line with most authors, investigations have shown that the immunosuppressant tacrolimus
is related to an increase in the number of nerve fibers and in axonal diameter, in
which axons could regenerate into various sizes and shapes.[4 ]
[17 ]
[21 ]
[35 ]
[38 ]
[39 ]
It was also found that tacrolimus is considered an immunosuppressant with greater
potency than cyclosporine; this result exposes the relationship between the use of
these drugs with the number of myelinated axons.[17 ]
[19 ]
[21 ]
[40 ]
Drug doses also show a relationship with the occurrence of their effects, although
most results have shown that there is no dose-dependent effect based on the use of
immunosuppressants.[4 ]
[10 ]
[21 ]
[37 ]
[39 ]
Regarding the mechanism of action of nerve regeneration, the authors mention that
the binding protein FKBP52 is responsible for the neurotrophic action of tacrolimus.
There is a high expression of GAP-43, an essential protein for the formation of a
growth cone and axonal elongation, in the presence of this drug in peripheral nerve
injuries, causing this protein to be maintained or to have its expression reinduced.[21 ]
[37 ]
[40 ]
Furthermore, the authors also highlighted the importance of using immunosuppressants,
especially when the lesions were repaired with allografts, because tacrolimus significantly
increases the rate of regeneration and its removal led to graft rejection, pronounced
functional deterioration, and loss of fibers in regeneration.[10 ]
[35 ]
Regarding myelin thickness and muscle weight, the authors found that tacrolimus doubled
the number of regenerated motoneurons and the thickness of the myelin sheath. In addition,
it promoted an increase in the rate of muscle weight recovery.[4 ]
[21 ]
[28 ]
[39 ]
[41 ]
Regarding sciatic function, tacrolimus has been shown to be effective in recovering
the sciatic functional index, although some authors have shown that these effects
would occur incompletely.[4 ]
[36 ]
[42 ]
[43 ]
Another factor to be emphasized are the adverse effects caused by tacrolimus and that
were mentioned by the authors, such as weight gain, superficial lesions at the injection
site, pruritus, nephrotoxicity or even death of animals.[10 ]
[19 ]
[28 ] This evidence reiterates the importance of using minimal doses of the drug to avoid
these adverse effects, as the efficacy of the drug does not show a close relationship
with the dose.
It is important to highlight the issues related to the risk of bias, considering the
methodological quality of the included studies, selection, and confounding variables.
Regarding structural and methodological aspects of the analysed texts, there was a
lack of information in the following categories: abstract, objectives, ethical procedures,
and collection procedures. The lack of information in these categories demonstrates
a structural and methodological deficiency in the developed studies, resulting in
incomplete work.
Furthermore, the noninclusion of other databases in the search for articles and publication
bias, since investigations with negative results are most often not published or are
published in journals not indexed in the selected databases, may interfere with generalization
of the results of the present review.
Another important factor refers to the existence of few studies correlated to nerve
regeneration procedures with grafts, and it is not possible to compare each nerve
injury technique. The present review found a lack of standardization of anaesthetic
substances, of doses of immunosuppressants, and of type of lesion. Thus, it is likely
that there are misinterpretations, although this was an alternative to include and
compare data from studies.
The limited number of studies and the methodological differences between them make
it difficult to generalize the results. Furthermore, most of the studies included
in this meta-analysis showed cross-sectional designs, making it difficult to analyse
the causal relationship between the use of immunosuppressants and peripheral nerve
regeneration. However, although it had many limitations, the results of the present
study are consistent.
Regarding the high heterogeneity of studies in general, a possible justification for
the relative variability of the results of experimental studies on nerve injuries
is the variety of models and test methods used. Besides, there was no time nor language
limitation, nor geographic aspects in the search for articles, which may influence
in some way.
It should also be mentioned that the search strategy was carried out by only one researcher,
which may contribute to some bias or loss of information/study during the execution
of the process. Not all articles included provided the mean, standard deviation, or
standard error values of the analysed variables; therefore, it is not possible to
calculate the effect size for all investigations. It was also observed that the sample
size was relatively small, favouring the development of type I errors, directly affecting
the effect size value of the studied variables.
Final Considerations
The present study aimed to carry out a systematic review with meta-analysis of studies
that address the issue of the repercussions of the pharmacological therapy of tacrolimus
and cyclosporine in the regeneration of peripheral nerves, seeking to compile the
results of studies that have been developed in recent years on the subject.
It was found that the effects of immunosuppressants on nerve regeneration were consistent,
mainly due to the increase in the number of myelinated axons in general, in the myelin
thickness, in the muscle weight, and in the sciatic functional index, regardless of
the dose administered.
We hope that the present study can contribute to scientific knowledge and applications
in the health context. In order to improve future research, authors of controlled
studies should opt for a more robust sampling approach, improving the matching of
the control group, because some variables can help to minimize differences between
groups; to validate indirect protocols for assessing peripheral nerve damage; to provide
more clarity on possible chronic complications that may compromise the neuronal function
of those involved, improving the characterization of the sample; and to avoid underestimating
or overestimating the experimental group or the control group.
