Ioannis Milonas

Transplanted neural precursor cells reduce brain inflammation to attenuate chronic experimental autoimmune encephalomyelitis.

Stem cell transplantation was introduced as a mean of cell replacement therapy, but the mechanism by which it confers clinical improvement in experimental models of neurological diseases is not clear. Here, we transplanted neural precursor cells (NPCs) into the ventricles of mice at day 6 after induction of chronic experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Transplanted cells migrated into white matter tracts and attenuated the clinical course of disease. NPC transplantation down-regulated the inflammatory brain process at the acute phase of disease, as indicated by a reduction in the number of perivascular infiltrates and of brain CD3+ T cells, an increase in the number and proportion of regulatory T cells and a reduction in the expression of ICAM-1 and LFA-1 in the brain. Demyelination and acute axonal injury in this model are considered to result mainly from the acute inflammatory process and correlate well with the chronic neurological residua. In consequence to inhibition of brain inflammation, precursor cell transplantation attenuated the primary demyelinating process and reduced the acute axonal injury. As a result, the size of demyelinated areas and extent of chronic axonal pathology were reduced in the transplanted brains. We suggest that the beneficial effect of transplanted NPCs in chronic EAE is mediated, in part, by decreasing brain inflammation and reducing tissue injury.

Axonal damage in multiple sclerosis: a complex issue in a complex disease.

Multiple sclerosis is no longer considered to simply be an autoimmune demyelinating disease. Axonal destruction is another central pathological feature and a contributor to the accumulating disability of disease progression. The mechanism underlying axonal pathology has not been fully clarified but does not appear to be a simple one. The relationship between axonal damage and other components of the pathological features such as demyelination, inflammation and remyelination are under intense investigation. Experimental data suggest that therapeutic interventions such as the induction of rapid remyelination may lead to the protection of axons. In addition to immunomodulation, future strategies for neuroprotection may be of great importance.

Neuroinflammation in multiple sclerosis: evidence for autoimmune dysregulation, not simple autoimmune reaction.

Both inflammatory and neurodegenerative components may contribute to the clinical profile of multiple sclerosis (MS) leading to irreversible deficits when they exceed the threshold of compensation. The mechanisms leading to tissue injury in MS are complex. Inflammation appears to be caused by overactive pro-inflammatory T-helper 1 cells, initiating an inflammatory cascade with several cellular and molecular immune components participating in the pathogenetic mechanism. Current treatments are most effective in the inflammatory phase of the disease since they may interfere with various stages of the immune cascade. Recent evidence has emerged that inflammation may not only be destructive, but may also play a part in tissue repair. This has opened up a new aspect of our knowledge of the role of the inflammatory process in MS. Data regarding the role of regulatory cells in particular, imply that specific immunomodulatory strategies that support the function of these particular cellular subpopulations may participate in the downregulation of autoimmune responses in MS.

Effectiveness of a new modified intraluminal suture for temporary middle cerebral artery occlusion in rats of various weight.

Intraluminal temporary middle cerebral artery occlusion (MCAO) is a common model of ischemic stroke in the rat with significant, suture and weight-dependent variability along with increased risk of subarachnoid haemorrhage (SAH). Our purpose was to increase reproducibility and decrease SAH using a modification of the Koizumi suture. We compared a Koizumi 5/0 Ethilon poly-l-lysine-coated suture (s-2, group B) to an identical, uncoated one (s-1, group A) and the Belayevs 3/0 suture (s-3, group C), in the 2-h MCAO model in Wistar rats of varying weight (310-527 g). Assessment included successful infarction rates, the modified neurological stroke scale (mNSS), a modified Bedersons scale (mBS), the grid-walking test (GWT), infarction volume (with rostrocaudal subanalysis and analysis of cortical/striatal involvement) and hemispheric edema. The s-2 suture increased the successful MCAO from 61.1% and 66.6% (groups A and C) to 97.5% in group B and induced a more severe clinical stroke (P<0.05) irrespective of animals weight, with no incidence of SAH. Infarction volume and ipsilateral hemispheric edema significantly (P<0.05) increased and well correlated with the mNSS (P<or=0.005), the mBS (P<or=0.01) and GWT outcomes. Our data suggest that the new modified suture induces a more reproducible ischemic stroke in Wistar rats for temporary-MCAO experiments, overcoming the variability of weight and the risk of SAH.

Epidemiological data of multiple sclerosis in the province of Evros, Greece.

The frequency of multiple sclerosis (MS) in Greece is still debated. Our previous epidemiological field survey with a cross-check study of MS on March 31, 1984, in the province of Evros in north-eastern Greece showed a prevalence rate of 10.1/100,000. In 1990, Milonas et al. recorded a prevalence rate of 29.5/100,000 in northern Greece. So Greece is classified in the medium-frequency zone according to Kurtzke. This study was performed to estimate the prevalence of MS in the province of Evros and the annual incidence rates from 1974 to 1999. Patients were identified from several sources. A clinical follow-up was performed in 95% of the cases, and, if clinically indicated, new paraclinical examinations were performed and cases classified by Poser’s criteria. The prevalence rate of the definite MS cases on December 31, 1999, was 38.9/100,000 and places the area in the high-risk zone. The mean annual incidence measured in 5-year intervals increased from 0.66/100,000 in 1974–1978 to 2.36/100,000 in 1994–1999 (p < 0.01). The increase in prevalence can be attributed to other causes than etiological changes, but the increase in the annual incidence rate indicates the possibility of a variation in risk factors of the disease.

A new model of subarachnoid hemorrhage in experimental animals with the purpose to examine cerebral vasospasm

Using 20 rabbits, we tried to establish a new model of experimental subarachnoid hemorrhage (SAH) for examining both acute and chronic cerebral vasospasm. A cranial opening was drilled, and a puncture made on the posterior branch of the middle cerebral artery. A second puncture was made in the superior sagittal sinus for additional withdrawal of subarachnoid blood. The bleeding thus induced resulted in arterial spasm which was studied by using serial electrocorticograms, cerebral blood flow measurement with 133Xe, and videomicroscopy of the small pial vessels at various intervals. After death of the animals, the brains were observed to identify the extention of the bleeding. It was indeed obvious that large amounts of subarachnoid blood clots had accumulated. This investigation showed that the rabbit can be used as a new experimental model of SAH. With a two-puncture method, it is possible to simulate the clinical phenomenon of a ruptured aneurysm, that seems to produce acute and chronic cerebral vasospasm. For the latter, the accumulation of blood clots in the basal surfaces plays an important role. The three methods of observation, videomicroscopy, cerebral blood flow measurements, and electrocorticography appeared to provide useful information in the study of biphasic vasospasm in the rabbit.

Amyotrophic lateral sclerosis: an introduction

Abstract Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease that results in the degeneration of lower and upper motor neurones in the brain and the spinal cord. Early onset and modern therapies, including assisted ventilation, improve survival in this disease, although its cause remains uncertain. Amongst the possible causes are deficiency of nerve growth factor, deficient glutamate re-uptake, autoimmunity and mutation of superoxide dismutase 1 gene. Additional factors may be industrial pollutants and occupational exposure to chemicals associated with welding and soldering. The criteria for the diagnosis of ALS, proposed by the World Federation of Neurology, are presented together with a review of the clinical features of the disease.

Inclusion body myositis in a patient with a presumed diagnosis of post-polio syndrome

Sirs: Sporadic inclusion body myositis (IBM) is a distinctive form of inflammatory myopathy and an important cause of progressive muscle wasting and weakness in later life [7]. Post-polio syndrome is defined as “the development of new muscle weakness and fatigue in skeletal or bulbar muscles, unrelated to any known medical cause, that begins 25–30 years after an acute attack of paralytic poliomyelitis” [4]. Searching the literature we found only two cases of pathologically confirmed inclusion body myositis in patients with a known history of paralytic poliomyelitis [1, 9]. In this letter we describe a similar case and discuss the data, which can lead to the reliable differential diagnosis between inclusion body myositis and postpoliomyelitis muscular atrophy. A farmer, aged 61 years, was referred to our department because of painless muscular weakness in his lower legs and right arm; his left arm had been severely paretic and atrophic since the age of 3 years following an acute attack of paralytic poliomyelitis. The new symptoms had started six months ago and there was progressive disability. At the time of the referral the patient could not rise from a chair and climb stairs without support. Neurological examination revealed muscular weakness mainly localized in the quadriceps femoris muscles (Medical Research Council Grade 2) and tibialis anterior in the lower limbs bilaterally, as well as in the deltoid, biceps brachii and deep finger flexor in the right arm (MRC Grade 3). Muscle tone and tendon reflexes were diffusely depressed. Severe atrophy was obvious in the quadriceps femoris. Sensory examination proved normal and the Babinski sign was not elicited. Routine laboratory tests showed mild elevation of creatine kinase (CK). The immunological profile of the patient was not normal; there were polyclonal hyperglobulinemia, positive titer of antinuclear antibodies (ANA) and low values of C4 complement indicating an autoimmune disorder. However, after an extensive review of the laboratory tests, the patient did not meet the criteria of any autoimmune disease. Needle electromyography of proximal (e. g. deltoid, vastus medialis) and distal muscles (tibialis anterior) of the right upper and the lower limbs revealed abnormal spontaneous activity, consisting of numerous fibrillation potentials and trains of positive sharp waves; additionally there was an excess of small amplitude and short duration polyphasic motor unit action potentials. Recruitment pattern was full with low amplitude potentials, indicating some kind of myopathic disorder. The electrophysiological findings in the left upper limb were compatible with a longstanding neurogenic disorder. An open muscle biopsy was performed on the right quadriceps (vastus medialis) muscle. In multiple frozen as well as paraffin sections of the skeletal muscle, a dense inflammatory infiltration and fibrosis of the endomysium was profound (Fig. 1). The inflammatory cells were mainly lymphocytes and most of them were T cells (Fig. 2). A variation of fiber size with large and small rounded fibers as well as degenerated fibers with or without phagocytosis was frequent. Other myopathic features, such as internal nuclei, splitting or ring fibers were also found. In some of the larger ones, phagocytosis without necrosis was obvious (Fig. 3). In a small number of muscle fibers one or more vacuoles with granular material were easily recognizable. This granular material was stained red by trichrome Gomori (Fig. 4). There were no signs of denervation or reinnervation. The morphology of this muscle biopsy specimen was consistent with inclusion body myositis. Unfortunately, an electron microscopic study of the muscle specimen was not performed. Treatment with prednisolone per os (0,75 mg/kg/day) was started tapering to a low maintenance dose after a period of 3 to 6 weeks combined with azathioprine 150 mg daily. In the follow-up after 3 months the assesment of muscle function in selected upper and lower limb muscle groups showed no further deterioration of the muscle strength. We believe that our patient suffered from inclusion body myositis and not from post-poliomyelitis muscle atrophy (PPMA) as originally was thought. At first, the selective pattern of muscle involvement in both the upper and lower limbs and especially the finding of knee extensor weakness that exceeded hip flexor weakness as well as finger flexor weakness were highly suggestive of inclusion body myositis. Our patient had also a progression of his symptoms noticeable in months, which is not consistent with the extremely slow course of PPMA. Moreover, new post-polio symptoms appear to begin after a mean period of approximately 30 years (range, 15 to 55) after the original attack of acute LETTER TO THE EDITORS

Evaluation of the Stability of Blood Flow over Time in the Dominant Hemisphere: A Functional Transcranial Doppler Study

Functional transcranial Doppler (fTCD) has been used for the identification of cerebral hemispheric dominance in various cognitive tasks. In our study, we have used fTCD with the aim to compare blood flow patterns in the hemispheres not only during the task activation periods but also in the post-stimulus phase. Normal volunteers, 25 right and 25 left-handed, were included. Mean flow velocities (FVs) in the bilateral middle cerebral arteries were recorded during the performance of six cognitive tasks and during the intervals between tasks. The lateralization index (LI) was calculated separately for each test (LI1-6), on the basis of the percent change of blood FV from baseline. To estimate flow fluctuations, a novel index, the LI-variability, was also calculated using a formula constituted by the minimum and maximum mean values recorded at specific time intervals during the entire procedure. Laterization indices, LI-3 and LI-4, corresponding to word generation and reading aloud tasks, produced the highest degree of activation. A perfect agreement (Cohens κ = 1.000, P < 0.001) was observed among LI-3, LI-4, and LI-V. The repetition of recordings gave excellent test—retest reliability in 10 randomly selected participants. Our results suggest that the hemisphere that is characterized as dominant by fTCD maintains a more stable flow pattern during the performance of successive cognitive tasks. Although it could not be considered as a clinically useful tool as yet, this observation introduces a novel parameter such as the stability of blood flow over time, which could potentially provide insight in the study of cerebral functions.

Animal models of central nervous system immune-mediated diseases: therapeutic interventions with bioactive peptides and mimetics.

Experimental allergic encephalomyelitis (EAE) is a T helper 1 (Th1) mediated autoimmune disease and the principal animal model for multiple sclerosis (MS). Like MS, EAE is characterized by a coordinated inflammatory attack on the myelin sheath in the central nervous system (CNS), with damage to axons. No matter whether the ideal animal model is not yet available, much knowledge concerning the pathogenesis of MS has been achieved through studies on EAE. Dissecting the underlying immune mechanisms provided recognition of several myelin antigens that are vulnerable in autoimmune attack. The beneficial effect and the mechanism of action of a number of the currently used immunomodulating agents in MS therapy were first indicated in EAE. Altered peptide ligands (APL) can modulate T-cell responses to native peptide antigens implicated in the pathogenesis of autoimmune diseases such as MS and EAE. However, peptide therapy is hindered due to the sensitivity of peptides to proteolytic enzymes as well as due to some immune-mediated side effects. A number of cyclic myelin peptide analogs seem to be potential candidates in maintaining the biological function of the original peptide and effective in controlling inflammation in EAE. Additional data regarding the immunomodulating and neuroprotective effect of these much promising agents is required. Based on the data from studies on EAE models, clinical trials should also be designed in order to elucidate the impact of such APL-induced immune responses in MS disease activity. These clinical trials should carefully incorporate monitoring of both clinical, neuroimaging and immunological parameters.