Pharmacology and therapeutics

Sensory improvement of leprosy peripheral neuropathy in

patients treated with perineural injection of platelet-rich
plasma
Sukma Anjayani1, MD, Yohanes Widodo Wirohadidjojo2, MD, Andi Muhammad Adam1,
3 4 1
MD, Danny Suwandi , MD, PhD, Arifin Seweng , MD, PhD, and Muh. Dali Amiruddin ,
MD, PhD

1
Department of Dermatology and Abstract
Venereology, Dr Wahidin Sudirohusodo Background Leprosy (Hansen’s disease) is a chronic granulomatous infection caused by
Hospital/Faculty of Medicine, Hasanuddin
Mycobacterium leprae with peripheral neuropathy as cutaneous and neurological
University, Makassar, Indonesia,
2
Department of Dermatology and
manifestations. Peripheral nerve regeneration may be stimulated by vascular endothelial
Venereology, Sardjito Hospital/Faculty of growth factor and other growth factors (GFs) that have important roles in extracellular
Medicine, Gadjah Mada University, matrix regeneration. All of those GFs can be found in platelet-rich plasma (PRP)
Yogyakarta, Indonesia, 3Department of preparation. The effect of PRP injection in leprosy peripheral neuropathy has never been
Pharmacology, Faculty of Medicine,
reported.
Hasanuddin University, Makassar,
Materials and methods A double-blind, randomized, control clinical trial was conducted
Indonesia, and 4Faculty of Public Health,
Hasanuddin University, Makassar, among 60 patients with leprosy peripheral neuropathy. They were randomized to receive
Indonesia either a 1-ml injection of PRP as treatment or a 1-ml injection of platelet-poor plasma
(PPP) as control. Skin sensibilities were measured by two-point discrimination test (TPDT)
Correspondence
and visual analog scale (VAS), which were taken before and two weeks after treatment.
Sukma Anjayani, MD
Department of Dermatology and
Results Perineural injection of PRP was shown to be significantly more effective than PPP
Venereology (P < 0.05) either in VAS or TPDT measurements. In both groups, the patients had a
Dr Wahidin Sudirohusodo Hospital/Faculty tingling sensation at the time of injection that disappeared shortly after.
of Medicine Conclusion This study shows that perineural PRP injection could promote improvement of
Hasanuddin University, Makassar,
peripheral neuropathy sensibility in patients with leprosy. More research is needed to better
Indonesia
E-mail: cukeyponk@gmail.com
determine the effects of PRP in nerve regeneration.

Conflict of interest: There is no conflict of

interest in terms of this research.

non-immunological, depending on the nature and charac-

Introduction
Leprosy is a chronic granulomatous disease caused by
Mycobacterium leprae, which has an affinity for periph-
eral nerves,1,2 and may affect sensory, motor, and auto-
nomic fibers. The sensory loss occurs earliest and is the
most frequently found among affected patients, but motor
loss can also occur.3 The most commonly involved nerves
are the posterior tibial, ulnar, median, lateral popliteal,
and facial nerves. Impaired sensation leads to trauma and
secondary infection, which may cause tissue damage and
deformities.1,2,4
The mechanisms of nerve damage in the patients
with neuritis leprosy could be either immunological or
teristics of the infiltrates.5 Among tuberculoid leprosy, the
high cell-mediated immune response and granulomatous
inflammation of the epineuron of peripheral nerves may
cause nerve thickness and rigidities resulting in nerve
damage under pressure of affected areas; while among
lepromatous leprosy, intraneural and extrafascicular gran-
ulomatous inflammation combined with bacterial prolifer-
ation within the Schwann cells leads to axonal atrophy.6
The reduction in axonal caliber and paranodal demyelina-
tion seen in leprosy nerves may result from dephosphory-
lation of neurofilament.7
Differing from central braches, which are well known
to be difficult to regenerate, periphery nerves have a
109

ª 2013 The International Society of Dermatology International Journal of Dermatology 2014, 53, 109–113
110 Pharmacology and therapeutics Sensory improvement of leprosy peripheral neuropathy with injection of PRP Anjayani et al.

better chance of regeneration.8 Based on Gardiner’s

review, in axonal regeneration, integrin and extracellular
matrix act as key mediators.9,10 Study among rat sciatic
nerves shows that increased vascularization may enhance
axonal regeneration, as was found among nerve defects
that were bridged with a silicone chamber containing vas-
cular endothelial growth factor (VEGF).10 VEGF and
other growth factors (GFs) for stimulating extracellular
matrix synthesis can be obtained from platelet-rich
plasma (PRP).11–13 In their review, Yu et al.14 and Rai-
mondo et al.15 suggest that GFs released from PRP may
promote regeneration of injured peripheral nerves. Here,
we reported the effect of PRP in stimulating improvement
of sensory nerves in patients with leprosy.

Materials and methods

Subjects
Sample size was calculated based on the hypothesis test for
two proportional studies with 80% of power statistic; the
calculated minimal sample size was 18 per group. After
obtaining approval from the local ethics committee of health and
medical research, 60 subjects who fulfilled study criteria were
enrolled in the study. The inclusion criteria were: (i) patient with
leprosy with peripheral neuropathy and hypoesthesia/
anesthesia; (ii) > 20 years old; (iii) hemoglobin  10 mg/dl
and platelet  100 000/mm3. The exclusion criteria were: (i)
peripheral neuropathy with other complaints; (ii) receiving
steroids one month prior to the study procedures; and
(iii) diabetes mellitus.
Design
All of the selected subjects were randomly allocated into two
groups: the first group received a 1-ml injection of PRP; and the
second group received a 1-ml injection of platelet-poor plasma
(PPP) as the placebo. The research design is shown in Figure 1.
PRP preparation
The PRP was produced based on the method of Krasna et al.16
Whole blood (5 ml) was taken from the cubital vein of each
patient, mixed well with 600 ll citrate dextrose as anticoagulant,
and centrifuged for seven minutes at 280 g at room
temperature. The plasma was decanted up to the erythrocyte
sediment and then centrifuged again for seven minutes at
1290 g at room temperature. Half of the supernatant plasma as
PPP was then aspirated and used as placebo. The rest of the
plasma and platelets were mixed well in order to produce PRP.
Experiments
Either PRP or placebo injections were performed in the posterior
tibial nerves and ulnar nerve. Before injection, the posterior tibial
nerve was found by palpation on the upper posterior medial
malleolus, and perineural injection was performed at 90° under
Figure 1 Research design. PRP, platelet-rich plasma; TPDT,
two-point discrimination test; VAS, visual analog scale
guidance of the physician’s fingers. The ulnar nerve was found
by palpation on the sulcus nervi ulnaris, between the medial
epicondylus and olecranon. Perineural injection was performed
at 45° under guidance of the physician’s finger. Sensory
functions of the peripheral nerves were assessed by the two-
point discrimination test (TPDT) and visual analog scale (VAS) at
baseline and two weeks after treatment on the same ink-marked
skin point and surrounding area. The efficacy of both treatments
was evaluated by comparing the mean of the TPDT and VAS
between both groups.
Statistical analysis
The following tests were performed: Chi-square for gender
variable; independent t-test for normal distribution variables or
Mann–Whitney for rejected normal distribution variables
between placebo and the PRP group; paired t-test for normal
distribution variables or Wilcoxon rank-signed test for rejected
normal distribution variables between baseline data and week 2
data. The level of significance was P < 0.05.
Results
After two weeks of the study, 60 patients were enrolled
in this experiment. Their characteristics are shown in
Table 1.
Based on Table 1, the patients characteristics between
PRP and the placebo group were equal (P > 0.05). The
TPDT either at baseline or in week 2 is shown in Figure 2.
Here, it can be observed that the TPDT at baseline was
equal (P> 0.05) between both groups. After receiving

International Journal of Dermatology 2014, 53, 109–113 ª 2013 The International Society of Dermatology
Anjayani et al. Sensory improvement of leprosy peripheral neuropathy with injection of PRP Pharmacology and therapeutics 111

Table 1 Patient characteristics Our study showed that the perineural injection of PRP
was able to stimulate nerve regeneration, as found in
Characteristics PRP n = 30 Placebo n = 30 Statistic improvement of the TPDT (Fig. 1) and in pain perception
by VAS analysis (Fig. 2). After injection, concentrated
Age (years, mean) 44.83  10.63 45  11.15 P = 0.953
Females 20 19 P = 0.787
platelets might be activated by collagen, such as previ-
Males 10 11 – ously reported by Jarvis et al.20 The collagen existed in
Hemoglobin levels 13.87  3.22 13.74  3.01 P = 0.875 neural and perineural tissues where the PRP injection was
Blood sugar levels 101.00  6.19 101.63  5.54 P = 0.678 performed. After being activated, platelets might release
Platelet levels 171.933  47.840 171.000  48.532 P = 0.940
various GFs, such as platelet-derived growth factor
(PDGF), transforming growth factor (TGF)-b1, VEGF, b-
PRP, platelet-rich plasma.
fibroblast growth factor, insulin-like growth factor, and
perineural injection, the TPDT of the PRP group showed epidermal growth factor.20 PDGF-BB has been known to
a significant reduction (P < 0.05) compared with the be capable of stimulating hyaluronan21 and glycosamino-
baseline, while among the placebo group this phenome- glicans22,23 as components of the extracellular matrix.
non was not found. As previously mentioned above, axon damage in the
The effect of PRP injection on the patients’ pain sensa- peripheral nerves had a better chance of being regener-
tion is shown in Figure 3. ated when compared with the central branches,8 and the
Here, it was observed that VAS at baseline was equal key mediators for this purpose are integrin and extracellu-
between the placebo and PRP groups. PRP injection lar matrix.9 Helmar and Ahmet24 in their review sug-
might stimulate the patients’ pain sensation measured by gested that Schwann cells are the therapeutic target in the
VAS in week 2. The significant VAS improvement treatment of peripheral neuropathy. This occurs because
(P < 0.05) was observed among the PRP group, while the Schwann cells express axon growth-promoting surface
placebo group showed no significant improvement. cell adhesion molecules and assemble the basal lamina as
a prerequisite for formation of myelin. In the processes of
myelin formation by Schwann cells, integrin has an
Discussion
important role in GF-mediated signaling.24 Deletion of
In order to obtain neurological parameters of the finely the integrin gene in Schwann cells leads to delayed
innervated area of skin, the TPDT was performed, with peripheral nerve regeneration, as found in the study of
sensitivity 75% and specificity 58%.2 The patients’ abil- sciatic nerve among mice with integrin b4-deleted gene.25
ity to discern that two nearby objects touching the skin Although there is no report on discovering the effect of
were truly two distinct points and not one was tested GFs in Schwann cell integrin gene expression, a study on
with two sharp points, and the length of the two dis- normal keratinocytes shows that integrin genes can be up-
cerned points reflected how finely innervated the area of regulated by TGF-b1.26,27
skin was.16–18 In addition, VAS was performed based on A study of sciatic nerves among mice has also shown
the patients’ choice of a horizontal line of 10 scales in that bridging nerves with silicone containing VEGF can
length for their own perception of pain of the ink- enhance sciatic nerve regeneration11; a similar event is
marked point stimulated by 10-Amp electrodesiccation. also reported in VEGF gene therapy.28
The more intense the pain, the longer the patients Our results indicated that TGF-b1 and VEGF contained
marked the horizontal line, as previously used in in PRP may be key factors in sensory impairment of those
Suswardana et al.’s19 report. leprosy peripheral neuropathy patients. Improvement of

Figure 2 Two-point discrimination test (TPDT): baseline and week 2. PRP, platelet-rich plasma

ª 2013 The International Society of Dermatology International Journal of Dermatology 2014, 53, 109–113
112 Pharmacology and therapeutics Sensory improvement of leprosy peripheral neuropathy with injection of PRP
Figure 3 Visual analog scale (VAS): baseline and week 2. PRP, platelet-rich plasma
sensory function is critical in prevention of tissue damage
and deformities among patients with leprosy. Because
PRP can easily be isolated from leprosy blood, injection
of PRP in the perineural-involved nerves may be effective
in preventing leprosy deformities. Further research into
longer observation times and more frequent injections is
necessary in order to support this suggestion.
Conclusion
PRP injection in perineural nerves of leprosy peripheral
neuropathy patients can improve peripheral nerve sensory
function in two weeks of observation. Further research
into longer observation times is necessary in order to sup-
port this suggestion.
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