INTRODUCTION

Tumor necrosis factor-alpha (TNFα) has toxic effects on myelin and endothelial cells^1,2. Matrix metallo-proteinases (MMPs) are endopeptidases implicated in tissue destruction and degradation of the blood-nerve barrier in CIDP³. Greater understanding of pathophysiology of CIDP may suggest the ideal way that facilitates diagnosis and treatment in these patients⁴. CIDP diagnosis is difficult because of heterogeneity of presentation and limitations of clinical, electrophysiologic diagnostic criteria⁵, TNF and MMP9 expression may be a helpful additional parameter for CIDP diagnosis. Current therapies for CIDP are ineffective in one-thirds of patient, fail to provide a durable clinical response and problematic for long-term therapy. Recently Etanercept; TNFα blocker; is tried to prevent inflammatory tissue damage in patients with CIDP and/or variants who are refractory or intolerant to conventional therapies⁶. Recently MMPs inhibitors were also found to attenuate clinical symptoms in CIDP and may be an improvement over the current approaches⁷. Nerve biopsy is considered useful for the diagnosis in most CIDP sets of criteria, and should be considered in patients in which the diagnosis is not completely clear or prior to initiating immunosuppressive therapy⁸. And it is mandatory for a definite diagnosis of CIDP in the American Academy of Neurology (AAN) criteria^9,10. There may be an added value of nerve biopsy in reaching a final diagnosis of CIDP which is the immunohistochemical assessment of MMP9 in nerve tissue^11,12.

PATIENTS AND METHODS

Patients:

This is a cross sectional case control study conducted on 60 patients; 42 male and 18 females; and 20 age and sex matched healthy control. The patients’ age ranged from 14 to 81 years old with a mean age 46.77±17.7 years were selected with clinical CIDP and the first three Electrophysiological criteria according to the European Federation of Neurological Societies/peripheral Nerve Society (EFNS/PNS) guideline on management of CIDP¹¹ and nerve conduction and electromyographic evidence of neuropathic affection with at least two months duration. The presence of the following were considered as exclusion criteria; drug or toxin exposure likely to  cause the neuropathy, hereditary demyelinating neuropathy, known or likely because of family history, foot deformity, mutilation of hands or feet, retinitis pigmentosa, ichthyosis, liability to pressure palsy, multifocal motor neuropathy.

Methods:

All patients were submitted to detailed history taking, general and neurological assessment

*       Modified Rankin Scale (MRS)¹³ and Neuropathy Impairment Score (NIS)¹⁴ were also performed for all patients.

MRS is a disability scales. Score ranged from 0-6 where 0 indicate no symptoms at all and 6 means death¹³. NIS provides objective scores of neurologic impairment. Sub scores of cranial nerves, muscle weakness upper and lower, reflexes and sensation were presented and the motor sub scores were divided into proximal and distal sets¹⁴.

*       Electrophysiologic evaluation: Motor and sensory nerve conduction studies (NCS) and EMG were performed by the standard methods. The normal values are referring to Lew and Tsai¹⁵. 

*       Tumor necrosis factor alpha: TNF-a EASIA was performed. The assay is based on an oligoclonal system. According to Misawa et al.¹⁶ TNF-a concentration was considered elevated if it was higher than 3 SD above the mean value of the control samples.

*       Sural nerve biopsies and Immunohistochemistry: Sections were prepared according to the standard method¹⁷. The specimen was divided into 2 parts: one part was processed for electron microscopic (EM) examination. The Immunohistological protocol was performed for the second part. Referring to Jan et al.¹⁸, the number of positive MMP-9 immunoreactive epineurial blood vessels were counted and given as number per total epineurial blood vessels in the cross section of the nerve; MMP9 reactive vessels ratio;.   

Statistical Analysis

The data were processed using SPSS v13. Chi-Square, Correlation coefficient and ANOVA tests were used.

RESULTS

Clinical Results

Most of our patients had gradual onset 48 patients (80%) and progressive course 50 patients (83.3%). The mean duration of the disease was 3.85±5.66 years. Mixed sensorimotor affection was present in the majority of patients 46 (76%), the minority had either pure motor affection 10 patients (17%) or pure sensory affection 4 patients (7%). Clinical data of the patients are presented in Table (1). Severity of the illness in the patients as indicated by means of NIS and MRS are presented in Table (2).

Neurophysiological results:

There was higher mean of distal latency and lower mean of amplitude and conduction velocity of the examined nerves of lower limbs compared to those of upper limbs,

Tumor necrosis factor alpha:

There was 44 patients (73.3%) having high level of TNF α (above the cutoff value; 31.7 pg/ml). The mean of TNF α levels in the patients group (50.13±41.24) was statistically significantly higher than that of the controls (12.5±6.19), (P<0.0001).

There was no statistical significant difference between the mean values of TNF α and different groups of gender, type of onset or course of CIDP.

Also there was no significant correlation between TNF α and age of the patients, age of onset, duration of illness and severity of illness; as indicated by NIS or MRS scores.

There was a positive correlation between TNFα and distal latency and a negative correlation between TNFα and conduction velocity of left ulnar nerve P<0.05 (Figure 1; Figure 2). There were no statistically significant correlations between TNF α levels and other items of nerve conduction studies.

There was no statistical significant difference between the mean value of TNFα and nerve biopsy items (P>0.05).

Sural nerve biopsy:

Demyelination was detected in all patients. Mild, moderate and severe demyelination was present in 26 patients (43.3%), 26 patients (43.3%) and 8 patients (13.3%) respectively. Presence of edema and regenerating clusters, endoneurial fibrosis and Schwann cells proliferation were evident in most of the patients (Figure 3; Figure 4). Table (3) summarizes the number and percent the patients having nerve biopsy abnormalities.

There was no statistically significant correlation between the items of nerve biopsy and the severity of illness.

The mean of conduction velocity of the peroneal nerve had a statistical significant difference between mild (I) and moderate (II) degree of demyelination in nerve biopsy (Table 4). There was no statistical significant difference between other NCS items and others nerve biopsy items P>0.05.

There was no statistical significant difference between the items of nerve biopsy and the mean value of TNFα or MMP9 reactive vessels ratio (P>0.05).

Matrix metalloproteinase (MMP9)

MMP9 reactive vessels were observed epineurially in 58 patients (Figure 5).

The mean of MMP9 reactive vessels ratio is 0.53±0.46 with a minimum 0 and a maximum 2.25.

There was no statistical significant difference between the mean value of MMP9 reactive vessels ratio and gender, onset or course of CIDP; P>0.05. There was no statistically significant correlation between MMP9 reactive vessels ratio and age, age of onset, disease duration or severity of illness as indicated by the total scores of NIS and MRS or NCS (P>0.05).

Table 1. Clinical presentation of the patients.

Table 2. Scores of NIS and MRS.

(MRS) Modified Rankin Scale.                                (NIS) Neuropathy Impairment Score

Table 3. Nerve biopsy items present in the patients.

Table 4. Comparison between conduction velocities of the examined nerves in different degree of demyelination.

*Significant

Figure 1. Correlation of tumor necrosis factor α (TNFα) and distal latency of left ulnar nerve.

Figure 2. Correlation of tumor necrosis factor α (TNFα) and conduction velocity of left ulnar Nerve.

Figure 5. Sural nerve biopsy from a male patient 70 years old with  CIDP of 8 years duration

showing positively stained epineurial blood vessels for MMP9 (®).

DISCUSSION

TNF can cause selective cytotoxic damage to human Schwann cells and myelinated fibers^19,20.²¹. In addition, TNF increase vascular permeability and breakdown of the blood–nerve barrier^22,23, contribute to pathogenesis of CIDP^24,25. In our study, there was statistically significant higher mean of TNFα in CIDP patients in comparison to that of controls. Although no significant correlations were found between TNF α levels and either the disease severity or the course of the disease, there was a positive correlation between TNFα levels and distal latency and negative correlation with conduction velocity of left ulnar nerve. This may point to importance of TNF in CIDP pathogenesis and diagnosis. Misawa et al.¹⁶ reported that high concentration of TNFα levels was associated with severe neurologic disability (using the Hughes functional grading scale), a relapsing course and longer distal latencies and slower conduction velocities in median, ulnar, and tibial nerve in NCS. Discrepancies between these results and ours can be explained by the small number of patients studied by these authors and the difference in the used methodology.

Nerve biopsy is considered useful for the diagnosis in most sets of criteria and it is mandatory for a definite diagnosis of CIDP in the AAN criteria focusing on demyelination and not inflammation⁹. The findings in nerve biopsy of our patients revealed mild to moderate degree of demyelination, Schwann cell proliferations edema, regenerating clusters and endoneurial fibrosis in most of the patients. Similarly Bosh & Smith²⁵, reported moderate reduction in myelinated fibers, edema, and segmental demyelination and remyelination in CIDP. Similarly Vallet²⁶ and S Kuwabara et al.²⁷ reported the presence of necrosis of the vessel walls and the endoneurial space and Schwann cell proliferations around remyelinating fibers or around the normally myelinated axon. But Toyka and Gold²⁸ and Chio et al.²⁹ described the presence of predominant demyelination and perivascular inflammatory infiltrates as the hallmarks of CIDP pathology. According to our results and others^25,26,27 demyelination and edema seems to be a more reliable finding for CIDP diagnosis than the presence of inflammatory cells. In this study there was no statistically significant correlation between items of nerve biopsy and the severity of illness, mean value of TNFα or MMP9 reactive vessels ratio (P>0.05). But the mean of conduction velocity of the peroneal nerve differs significantly between mild (I) and moderate (II) degree of demyelination in nerve biopsy. This may be explained by the patchy nature of the disease and as the sural nerve is a branch of the common peroneal nerve, the degree of demyelination in sural nerve was correlated to the conduction velocity of the peroneal nerve.

There may be an added value of nerve biopsy in reaching a final diagnosis of CIDP which is the immunohistochemical assessment of MMP9 in nerve tissue^30,31. Positive epineurial blood vessels for MMP9 were observed in 58 of our patients. This is in agreement with Renaud³², who proved that MMP9 expression was highest in the epineurium and specifically around the blood vessels which may be explained by the possibility that MMP9 derives essentially from blood derived immune cells.

In our study, no significant correlation was found between MMP9 reactive vessels percentage and severity of illness.

Accordingly, we can suggest that MMP9 may be considered a sensitive marker as it present in nearly all the cases but it was not correlated to the severity of the disease. This may be explained by the hypothesis that the clinical presentation and course of CIDP may not be correlated with the expression of this marker. Our results pointed that MMP9 expression may be a helpful additional parameter for the diagnosis of CIDP and that MMP9 inhibitors may be considered as a novel therapeutic approach in the future.

Conclusion and Recommendations

TNFα, MMP9 and the presence of demyelination or edema in sural nerve biopsy is an important parameter for the diagnosis of CIDP. Controlled therapeutic trials on anti TNFα and MMP9 inhibitor to evaluate them as new models of CIDP treatment is recommend.

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