SEARCH

SEARCH BY CITATION

Keywords:

  • carpal tunnel;
  • dextrose;
  • median nerve;
  • platelet-rich plasma;
  • rabbit

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Introduction: We evaluated the effect of platelet-rich plasma (PRP) injection in a rabbit model of dextrose-induced median nerve injury. Methods: New Zealand white rabbits (n = 15) were divided randomly into 3 groups. Three different regimens (group 1: 0.1 ml saline; group 2: 10% dextrose with PRP; group 3: 10% dextrose with saline) were injected within the carpal tunnel. Electrophysiological and histological findings were evaluated 12 weeks after the injection. Results: The mean median motor latency in group 3 was significantly longer than that in groups 1 and 2. The cross-sectional area of the median nerve and subsynovial connective tissue thickness in group 3 were significantly larger than those in groups 1 and 2. Conclusions: PRP injection may be effective in controlling median nerve injury, as demonstrated by improvement in electrophysiological and histological findings 12 weeks after dextrose injection. Muscle Nerve 49: 56–60, 2014

Abbreviations
CMAP

compound muscle action potential

CSA

cross-sectional area

CTS

carpal tunnel syndrome

PRP

platelet-rich plasma

SSCT

subsynovial connective tissue

Idiopathic carpal tunnel syndrome (CTS) is characterized by non-inflammatory fibrosis and thickening of the subsynovial connective tissue (SSCT) within the carpal canal.[1, 2] Previous studies have shown that fibrosis might lead to an increased volume within the carpal tunnel and thus cause nerve compression.[3, 4]

A recent systemic review reported that corticosteroid treatment decreased inflammation and edema, but there are possible side effects.[5] The most significant side effect is that it reduces collagen and proteoglycan synthesis and weakens the mechanical strength of the tendon, thereby resulting in further degeneration.[5] A previous study showed that autologous blood injection into the carpal tunnel of patients with refractory CTS improved pain intensity and electrodiagnostic parameters. The investigators suggested that autologous blood injection caused improvement in the regenerative process.[6]

Platelets contain an abundance of growth factors and cytokines that are crucial in the healing process of soft tissues and bone mineralization.[7] Platelets play an instrumental role in the normal healing response via local secretion of growth factors and recruitment of reparative cells.[7] Compared with whole blood, PRP is composed of an approximately 3–8-fold greater concentration of platelets and contains a hyperphysiological content of autologous growth factors. A recent study demonstrated that PRP injection seems to be superior to autologous blood injection for chronic or refractory lateral elbow epicondylitis.[8]

Considering the regenerative effects of PRP on connective tissue, we hypothesized that an injection of PRP into the carpal tunnel of rabbits that had undergone 10% hypertonic dextrose-induced nerve injury would improve healing and nerve regeneration, as evidenced by histological examination and electrophysiological testing. This study was designed to investigate this hypothesis in a rabbit model of dextrose-induced median nerve injury using ultrasound guidance to enhance the accuracy of the injections.

METHODS

  1. Top of page
  2. ABSTRACT
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Animals

Fifteen healthy, unexercised, male New Zealand white rabbits (weighing 4.0–4.5 kg) were used in this study, which was performed in accordance with internationally accredited guidelines and approved by the animal research ethics committee of Daegu Catholic University. All animals were housed in separate metal cages at 23 ± 2°C with 45 ± 10% relative humidity. They were provided free access to tap water and were fed a commercial rabbit diet. The animals were allowed ad libitum movement in their cages (approximately 65 cm × 45 cm × 30 cm).

Experimental Median Nerve Injury Model

Anesthesia was induced using isoflurane (JW Pharmaceutical, Goyang, Korea) vaporized in oxygen and delivered via a large animal cycling system. The 15 rabbits were divided randomly into 3 groups of 5 rabbits. The left forepaw of each rabbit was shaved and sterilized, and 3 different regimens were prepared for carpal tunnel injection. The control group (group 1) received 2 separate injections, 1 week apart, with 0.1 ml of saline. Group 2 and 3 rabbits received 2 separate injections, 1 week apart, with 0.1 ml of 10% dextrose. At 4 weeks after the first injection, groups 1 and 3 received an additional 0.3-ml saline injection, and group 2 received an additional 0.3-ml PRP injection. The rabbits were euthanized by carbon monoxide inhalation 12 weeks after the first injection. All injections were done using visualization with a commercially available ultrasound system E-CUBE 9 3–12-MHz multifrequency linear transducer (Alpionion Medical Systems, Seoul, Korea) (Fig. 1A). All procedures were done while animals were under general anesthesia with sterile conditions. No medication was administered after the injection.

image

Figure 1. Injection into subsynovial connective tissue in rabbit under ultrasound guidance (A) and median nerve conduction study in rabbit (B, C). N, needle (arrows); MN, median nerve; +, anodal electrode for stimulation; −, cathodal electrode for stimulation. R1 & R2, recording electrode on thenar muscles. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

PRP Preparation, Platelet Count Analyses, and Regimen Administration

For PRP preparation, 6.0 ml of venous blood was drawn from the marginal auricular vein (using aseptic technique) and mixed with 2 ml of 0.129 mol/L sodium citrate in an sPRP system PRP device (Huons, Goyang, Korea). The mixture was centrifuged for 3 minutes at 3200 rpm and divided into 3 layers that were comprised of platelet-poor plasma, PRP, and red blood cells. The PRP layer was extracted through a special port, and 0.3 ml was used for injection. The number of platelets from whole blood and the isolated PRP fraction were assessed using the scil Vet ABC Plus hematology analyzer (Scil Animal Care, Gurnee, Illinois). The mean platelet concentration of whole blood was 310 ± 24 × 103/μl (range 265–332 × 103/μl). The mean platelet concentration of the PRP fraction was 2464 ± 870 × 103/μl (range 1901–3010 × 103/μl), which represented an 8.0-fold increase over the whole blood concentration.

Electrophysiological Measurements

Electrophysiological study was performed under general anesthesia on the median nerve of the left forepaw. The compound muscle action potential (CMAP) latency was recorded from the thenar muscle at a distance of 3 cm (Fig. 1B and C). Skin temperature was measured using a digital needle thermometer and was maintained at 34°–36°C. Recordings were performed before injection, and at 4 and 12 weeks after the first injection.

Histological Analyses

All animals were euthanized at 12 weeks after the first injection. The front paws were then harvested, and the total contents of the carpal tunnel were divided and prepared. After primary fixation for 24 hours in 4% paraformaldehyde, the tissue selected for nerve histology was immersed in a fixative solution of 10% glutaraldehyde and 10% paraformaldehyde for secondary fixation. The tissue was embedded in plastic resin. Cross-sections (0.6 mm) of the median nerve were made at the carpal tunnel level and stained with hematoxylin and eosin. Sections were evaluated qualitatively for fascicular demyelination and subperineurial edema using a light microscope (BH-2; Olympus Co.). In addition, the cross-sectional area (CSA) of the median nerve and SSCT thickness were measured at the mid-carpal tunnel level. A physiatrist who was blinded to the treatments evaluated all sections.

Statistical Analyses

Statistical analyses were performed using SPSS software, version 19.0 (SPSS, Inc., Chicago, Illinois). The Wilcoxon signed-rank test was used to assess the difference in median CMAP latency between baseline and after injection in each group. The Kruskal–Wallis test was used to determine differences between the 3 groups in the parameters measured (median CMAP latency, CSA of median nerve, and SSCT thickness). P < 0.05 was considered statistically significant.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

In group 3, the mean median CMAP latency at 12 weeks was significantly longer than before injection and 4 weeks after the injection (Table 1; P < 0.05). However, there was no significant difference in mean median CMAP latency in groups 1 and 2 (Table 1).

Table 1. Electrophysiological findings after injection.
 Median compound muscle action potential latency (ms)
BaselineAfter 4 weeksAfter 12 weeks
  1. Values expressed as mean ± standard deviation. D, 10% dextrose; PRP, platelet-rich plasma.

  2. a

    P < 0.05 by Wilcoxon signed-rank test between baseline and after 12 weeks.

  3. b

    P < 0.05 by Wilcoxon signed-rank test between after 4 weeks and after 12 weeks.

Group 1 (saline)1.54 ± 0.151.55 ± 0.161.56 ± 0.13
Group 2 (D with PRP)1.56 ± 0.161.58 ± 0.181.78 ± 0.09
Group 3 (D with saline)1.55 ± 0.141.57 ± 0.172.17 ± 0.22ab

The mean median CMAP latency in group 3 was significantly longer than in groups 1 and 2 at 12 weeks after injection (Table 2; P < 0.05). Normal histology was observed in group 1 (Fig. 2A). There was interstitial and somewhat disorganized collagen deposition with fibrosis in group 2 (Fig. 2B), and demyelination of median nerve in association with interstitial organized fibrosis with collagen deposition and perineural fibrosis in group 3 12 weeks after injection (Fig. 2C). The mean CSA of the median nerve and the mean SSCT thickness in group 3 were significantly larger in groups 1 and 2 at 12 weeks after injection (Figs. 2 and 3 and Table 2; P < 0.05).

Table 2. Comparison of electrophysiological and histological findings among the 3 groups at 12 weeks post-injection.
 Group 1 (saline)Group 2 (D with PRP)Group 3 (D with saline)P
  1. Values expressed as mean ± standard deviation. CMAP, compound muscle action potential; CSA, cross-sectional area; SSCT, subsynovial connective tissue; D, 10% dextrose; PRP, platelet-rich plasma.

  2. a

    P < 0.05 by Kruskal–Wallis test among the 3 groups.

Median CMAP latency (ms)1.56 ± 0.131.78 ± 0.09a2.17 ± 0.22a0.003
Median nerve CSA (mm2)0.49 ± 0.120.98 ± 0.84a2.54 ± 1.64a0.038
SSCT thickness (mm)0.05 ± 0.020.13 ± 0.04a0.24 ± 0.06a0.008
image

Figure 2. Cross-sectional area (arrows) of median nerve was calculated at the mid-carpal tunnel level (hematoxylin–eosin stain; original magnification ×400). Group 1 appeared to have normal histology at 12 weeks (A). Interstitial, somewhat disorganized collagen deposition with fibrosis was observed in group 2 at 12 weeks (B). Demyelination of the median nerve was identified in association with interstitial fibrosis with collagen deposition and perineurial fibrosis in group 3 specimens at 12 weeks (C). FDS, flexor digitorum superficialis. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

image

Figure 3. Thickness of subsynovial connective tissue (arrows) between flexor digitorum superficialis (FDS) and flexor digitorum profundus (FDP) was measured between group 2 (A) and group 3 (B) at the mid-carpal tunnel level (hematoxylin–eosin staining; original magnification ×100). FDS, flexor digitorum superficialis; FDP, flexor digitorum profundus. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

We found that, compared with saline injection, PRP injection into the carpal tunnel of rabbits with dextrose-induced median nerve injury significantly reduced swelling of the median nerve, suggesting improved healing and potential for better nerve recovery, as shown by histological examination. However, electrophysiological findings did not correspond with the histological findings, particularly with regard to CSA of the median nerve and SSCT thickness in the carpal tunnel 12 weeks after injection. Mean CSA of the median nerve and SSCT thickness in group 2 were significantly greater in group 1 12 weeks after injection. In contrast, there was no significant difference in mean median CMAP latency between groups 1 and 2 12 weeks after injection. Thus, our hypothesis is only supported partially.

There are several possible explanations for this discrepancy. First, sensory fibers are more sensitive to compression than motor fibers in median nerve injury. Hence, a sensory nerve conduction study would be more sensitive in the diagnosis of CTS than a motor nerve conduction study. Sensory fibers are more susceptible to ischemic changes, considering the larger proportion of large myelinated fibers and higher energy requirements than motor fascicles. Therefore, sensory fibers are more likely to show early damage from compression.[9, 10] However, it is technically quite difficult to conduct median sensory nerve conduction studies in the rabbit model. Second, motor nerve changes are relatively late findings. This notion was supported by an earlier finding in patients with chronic symptoms and negative electrophysiological findings who underwent surgery. In all patients, histological changes, such as edema formation and perineural fibrosis, were observed as operative findings. Third, there is discordance between symptoms and electrophysiological study in approximately 16–34% of CTS patients.[11, 12] Therefore, ultrasound imaging is an emerging complementary diagnostic method in clinically suspected CTS cases when electrophysiological studies are normal.

This study shows that 2 injections of 10% dextrose under ultrasound guidance induced SSCT fibrosis, resulting in median nerve swelling and focal slowing of median motor nerve conduction. Our injection method was different from that used in a previous study using the same injection regimen; that study showed the effects of a double injection of 10% dextrose solution through operative dissection in the SSCT of the rabbit carpal tunnel on the morphology of the SSCT and median nerve.[13] Local injection into the rabbit carpal tunnel under ultrasound guidance has not been reported until now. In a human study, the performance, clinical outcome, and cost-effectiveness of injection of the carpal tunnel was significantly better under ultrasound guidance than conventional blind, palpation-guided injection.[14] In our study, the injection technique into the rabbit carpal tunnel under ultrasound guidance was similar to that used in human CTS.

The ideal concentration of platelets in PRP is not yet clear. One investigation demonstrated that the concentration of growth factors in PRP correlated positively with the platelet concentration of PRP.[15] Qualitative and quantitative platelet changes may affect the regenerative power of PRP. In our study, PRP was prepared by centrifugation of autologous whole blood, and the platelet concentration of whole blood was compatible with the normal value range for the rabbit (158–650 ×103/μl).[16] The platelet concentration of PRP was 8.0-fold higher than that of whole blood, thus offering higher regenerative effects. Therefore, our PRP preparation is adequate for assessing its therapeutic effects on median nerve regeneration because of the high platelet concentration.

General anesthetics (mainly halogenated volatile anesthetics) may inhibit neuronal voltage-gated Na+ channels in unmyelinated axons, inhibiting presynaptic action potentials as a result of Na+ channel blockade.[17] In our investigation, the electrophysiological study was performed under general anesthesia using isoflurane. However, isoflurane does not have significant effects on voltage-gated Na+ channels in large myelinated axons such as the median nerve.[17] Therefore, the electrophysiological study findings should have been unaffected by isoflurane inhalation.

There are several limitations to this study. First, the small number of animals made it difficult to determine the therapeutic effect of PRP in rabbit median nerve injury. Second, we did not determine whether PRP injection 12 weeks after hypertonic dextrose injection would have the same therapeutic effects in rabbit median nerve injury as those in chronic CTS in humans. Several studies have shown that PRP injection is an effective treatment for chronic musculoskeletal diseases.[18-20] The SSCT exhibited early changes, such as increased cellularity and evidence of vascular proliferation with collagen remodeling at 4 weeks after hypertonic dextrose injections.[13] We conducted PRP injection 4 weeks after hypertonic dextrose injection, because PRP may inhibit collagen remodeling and vascular proliferation in the SSCT of the carpal tunnel. The results show that early PRP injection may be effective in controlling disease progression. Third, we did not evaluate the therapeutic effects of PRP in severe median nerve injury. Severe median nerve injury would be indicated by prolonged distal median motor latency with low-amplitude median CMAP.[21] In a previous study,[13] the investigators evaluated a single rabbit 16 weeks after the first dextrose injection. The rabbit showed evidence of demyelination on transmission electron microscopy along with reduced CMAP amplitude and decreased motor nerve conduction. However, we found that the median CMAP latency had a significant delay at 12 weeks after hypertonic dextrose injection, and this finding was compatible with previous findings on moderate median nerve injury.[21, 22] We did not assess the median CMAP amplitude in our study, because measurement of CMAP amplitude using needle recording is not reliable due to interindividual variability.[23] Thus, further studies will be needed to determine the effects of PRP in rabbit median nerve injury using different platelet concentrations, injection times, number of injections, and severity levels of median nerve injury, with the goal of achieving the best and most durable outcomes. PRP has many beneficial effects on peripheral nerve regeneration after nerve injury by autologous supply of growth factors.

In conclusion, these results indicate that a 10% dextrose injection in the carpal tunnel of rabbits under ultrasound guidance can result in delayed median CMAP latency, SSCT thickening, and median nerve swelling; these findings are comparable to those observed in human CTS studies. PRP injection may be effective in controlling the progression of median nerve injury, as demonstrated by significant improvement in electrophysiological and histological findings 12 weeks after PRP injection in a rabbit model of dextrose-induced median nerve injury.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  • 1
    Ettema AM, Amadio PC, Zhao C, Wold LE, An KN. A histological and immunohistochemical study of the subsynovial connective tissue in idiopathic carpal tunnel syndrome. J Bone Joint Surg Am 2004;86:14581466.
  • 2
    Neal NC, McManners J, Stirling GA. Pathology of the flexor tendon sheath in the spontaneous carpal tunnel syndrome. J Hand Surg Br 1987;12:229232.
  • 3
    Gelberman RH, Hergenroeder PT, Hargens AR, Lundborg GN, Akeson WH. The carpal tunnel syndrome. A study of carpal canal pressures. J Bone Joint Surg Am 1981;63:380383.
  • 4
    Phalen GS. The carpal-tunnel syndrome. Seventeen years′ experience in diagnosis and treatment of six hundred fifty-four hands. J Bone Joint Surg Am 1966;48:211228.
  • 5
    Wong MW, Tang YN, Fu SC, Lee KM, Chan KM. Triamcinolone suppresses human tenocyte cellular activity and collagen synthesis. Clin Orthop Relat Res 2004;421:277281.
  • 6
    Jazayeri SM, Azizi S, Moghtaderi AR. Autologous blood injection in carpal tunnel syndrome (CTS). Electromyogr Clin Neurophysiol 2009;49:369372.
  • 7
    Kwon DR, Park GY, Lee SU. The effects of intra-articular platelet-rich plasma injection according to the severity of collagenase-induced knee osteoarthritis in a rabbit model. Ann Rehabil Med 2012;36:458465.
  • 8
    Thanasas C, Papadimitriou G, Charalambidis C, Paraskevopoulos I, Papanikolaou A. Platelet-rich plasma versus autologous whole blood for the treatment of chronic lateral elbow epicondylitis: a randomized controlled clinical trial. Am J Sports Med 2011;39:21302134.
  • 9
    Atroshi I, Gummesson C, Johnsson R, Ornstein E, Ranstam J, Rosén I. Prevalence of carpal tunnel syndrome in a general population. JAMA 1999;282:153158.
  • 10
    Werner RA, Andary M. Electrodiagnostic evaluation of carpal tunnel syndrome. Muscle Nerve 2011;44:597607.
  • 11
    Gomes I, Becker J, Ehlers JA, Nora DB. Prediction of the neurophysiological diagnosis of carpal tunnel syndrome from the demographic and clinical data. Clin Neurophysiol 2006;117:964971.
  • 12
    Witt JC, Hentz JG, Stevens JC. Carpal tunnel syndrome with normal nerve conduction studies. Muscle Nerve 2004;29:515522.
  • 13
    Oh S, Ettema AM, Zhao C, Zobitz ME, Wold LE, An KN, et al. Dextrose-induced subsynovial connective tissue fibrosis in the rabbit carpal tunnel: a potential model to study carpal tunnel syndrome? Hand (NY) 2008;3:3440.
  • 14
    Chavez-Chiang NR, Delea SL, Sibbitt WL Jr, Bankhurst AD, Norton H. Outcomes and cost-effectiveness of carpal tunnel injections using sonographic needle guidance [abstract]. Arthritis Rheum 2010;62(suppl):S677:1626.
  • 15
    Li M, Zhang C, Yuan T, Chen S, Lu R. Assessment study on a set of platelet-rich plasma preparation. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2011;25:112116.
  • 16
    Fox JG, Anderson LC, Loew FM, Quimby FW. Laboratory animal medicine, 2nd edition. Salt Lake City, UT: Academic Press; 2002.
  • 17
    Hemmings HC Jr. Sodium channels and the synaptic mechanisms of inhaled anaesthetics. Br J Anaesth 2009;103:6169.
  • 18
    Filardo G, Kon E, Della Villa S, Vincentelli F, Fornasari PM, Marcacci M. Use of platelet-rich plasma for the treatment of refractory jumper's knee. Int Orthop 2010;34:909915.
  • 19
    Mishra A, Pavelko T. Treatment of chronic elbow tendinosis with buffered platelet-rich plasma. Am J Sports Med 2006;34:17741778.
  • 20
    Kon E, Filardo G, Delcogliano M, Presti ML, Russo A, Bondi A, et al. Platelet-rich plasma: new clinical application: a pilot study for treatment of jumper's knee. Injury 2009;40:598603.
  • 21
    Mohammadi A, Ghasemi-Rad M, Mladkova-Suchy N, Ansari S. Correlation between the severity of carpal tunnel syndrome and color Doppler sonography findings. AJR Am J Roentgenol 2012;198:181184.
  • 22
    Yoshii Y, Zhao C, Schmelzer JD, Low PA, An KN, Amadio PC. The effects of hypertonic dextrose injection on connective tissue and nerve conduction through the rabbit carpal tunnel. Arch Phys Med Rehabil 2009;90:333339.
  • 23
    Alfonsi E, Moglia A, Sandrini G, Pisoni MR, Arrigo A. Electrophysiological study of long thoracic nerve conduction in normal subjects. Electromyogr Clin Neurophysiol 1986;26:6367.