Isoflurane

Background and methods: Immunomodulation with intravenous immunoglobulin (IVIG) represents a way of interfering with the disease process in multiple sclerosis (MS). In this study, the effects of IVIG on neurological symptoms and central nervous system (CNS) pathology were evaluated in experimental autoimmune encephalomyelitis (EAE), an MS animal model. EAE was induced in susceptible Dark Agouti rats by active immunization with a spinal cord homogenate, and infusions of 1 g/kg IVIG were given prophy/actically or therapeutically.

Results: The administration of IVIG at the time of immunization significantly suppressed the development of neurological symptoms compared with infusions of placebo (mean EAE score 0.6±0.3 versus 2.3±0.4). Moreover, the prophylactic IVIG administration resulted in a significant inhibition of the inflammatory response in CNS tissue (inflammation score 1.1±0.2 versus 1.8±0.2 after placebo). No beneficial effects were obtained by therapeutic IVIG infusions as the EAE disease course and the degree of inflammation and demyelination in the CNS were not different from animals receiving treatment with placebo.

Conclusions: The present study indicates that IVIG reduces the symptoms of EAE by suppression of the CNS inflammation that characterizes CNS pathology in these animals. Taking into account data from clinical trials of IVIG in MS, the results further suggest that IVIG acts primarily during the induction phase of the immune response thus preventing the development of relapses in MS. [Neural Res 2005; 27: 591-597]

Keywords: Intravenous immunoglobulin; immunoglobulin G; experimental autoimmune encephalomyelitis; multiple sclerosis; Dark Agouti rat

INTRODUCTION

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system. The pathology of MS is characterized by white matter lesions of focal demyelination and axonal damage in the brain and spinal cord1,2. Initially, most patients have a relapsingremitting disease course that typically shifts towards ongoing progression later, i.e. secondary progressive MS1. The cause of MS still has to be resolved, but the interaction of genetic predisposition and yet unidentified environmental factors are thought to be responsible3,4. Because of the lack of measurable trigger factors, the most important diagnostic criterion is the demonstration of dissemination of demyelinated central nervous system (CNS) lesions in both time and space, generally achieved by magnetic resonance imaging5. Pathologically, the hallmark of MS is the destruction of myelin and axonal degeneration driven by both T lymphocytes and antibodies2,6,7. Current therapies are only partially effective in attenuating disease activity, and MS is, therefore, still a disease with unmet medical needs.

Clinical trials have shown that immunoglobulin for intravenous administration (IVIG) has the potential to reduce the disease activity in MS8-10. However, the mechanisms by which IVIG may interfere with the pathophysiology of MS are not yet fully understood. The effects of IVIG treatment presumably owe to the variety of antibody specificities present in the IVIG preparations. IVIG may target harmful autoantibodies by anti-idiotypic interactions11,12, but may also inhibit inflammatory cells by binding through the constant Fc region of the immunoglobulin molecule13,14. Furthermore, IVIG may inhibit damage mediated by the complement system15,16 and may be involved in myelin repair through the process of remyelination17,18.

The classical animal model for studying MS, experimental autoimmune encephalomyelitis (EAE), is an inflammatory disease of the CNS primarily mediated by T cells19. EAE with a protracted and relapsing disease course resembling MS can be induced by the immunization of susceptible rat strains with CNS antigens20,21. Like MS, EAE in rodents may also be associated with axonal damage and neurodegeneration22,23. IVIG administration has previously been reported to significantly ameliorate the neurological symptoms of EAE24,25. In the present study, we induced EAE in the susceptible rat strain Dark Agouti (DA) in order to study the effects of IVIG treatment on the pathological changes in the CNS. The animals were treated with infusions of IVIG administered prophylactically or therapeutical Iy, and the CNS histopathology was studied during the acute attack at day 11 and 4 weeks after the induction of EAE.

MATERIALS AND METHODS

Animals

A protracted and relapsing type of EAE was induced in male rats of the susceptible inbred strain DA (180-200 g; Harlan, Horst, The Netherlands). The animals were acclimatized for a minimum of 5 days and maintained on a 12-hour light-dark cycle. Food and water were given ad libitum. The experiments were conducted according to the guidelines of the Danish Committee for Animal Experiments.

Induction of EAE

EAE was induced by subcutaneous inoculation of 0.2 ml encephalitogenic emulsion at the base of the tail. The emulsion was prepared as follows: spinal cord tissue obtained from normal male DA rats (>225 g) was homogenized in saline (1 g tissue/1 ml 0.9% NaCl, pH 7.4) and emulsified in an equal volume of incomplete Freund's adjuvant (IFA) (Difco Laboratories Inc., Detroit, Ml, USA). During the immunization, the animals were kept under light anaesthesia by isoflurane inhalation (Abbott Scandinavia AB, Solna, Sweden). The control rats were inoculated with saline.

Evaluation of EAE symptoms

Throughout the study, the animals were evaluated on a daily basis for weight loss and neurological symptoms of active disease. The EAE symptoms were assessed according to the following scoring system: 0, no clinical signs; 1, tail paralysis; 2, mild-to-moderate hind limb paresis; 3, severe hind limb paresis; 4, paralysis of the limbs; 5, tetraplegia; 6, moribund state or death from EAE.

IVIG administration

Human IVIG (10% IgG; Bayer AG, Leverkusen, Germany) was administered on 2 consecutive days: as prophylactic treatment at the time of EAE induction at day 0 and 1 post-immunization (p.L), or in a therapeutic treatment protocol at the beginning of the acute EAE attack at days 8 and 9 p.i. (EAE+ IVIG, n=12 per group). A dose of 1 g IgG/kg was administered into a tail vein at an infusion rate of 0.2 ml/minute. The control animals received an intravenous infusion of either placebo solution (0.1% albumin in 10% maltose; Bayer AG, Leverkusen, Germany) (EAE+ placebo, n=12 per group) or saline (0.9% NaCI, pH 7.4) (EAE control, n=7; saline control, n=7).

Histological evaluation

The experiments were stopped at two different time points: during the acute EAE attack at day 11 p.i., or during the remission of the disease at day 28 p.i.. The animals were anaesthetized with isoflurane and perfused intracardially with phosphate-buffered saline. The brain and spinal cord segments C^sub 5-7^ and L^sub 1-3^ were dissected and fixed in 4% paraformaldehyde. The tissues were then dehydrated, embedded in paraffin and 3-µm sections were cut using a microtome. Random numbers were assigned to the sections, and the study remained blinded until after the final evaluation. The sections were rehydrated through xylene and ethanol before staining with haematoxylin and eosin or Kluver-Barrera stain for the assessment of inflammation and demyelination. The degree of inflammatory changes in the CNS was evaluated according to the following scoring system: grade 0, no inflammation; grade 1, minor perivascular infiltration; grade 2, several perivascular and parenchymal infiltrations; grade 3, maximal inflammatory response and demyelination.

Statistical analysis

Data are presented as mean±SEM. The Student's t-test was used for the comparison of changes in body weight. Clinical and histological scores were compared for significant differences between the treatment groups by the Mann-Whitney rank sum test. The Spearman rank order correlation was used to determine the association between the clinical or histological scoring and body weight changes. Probability values below 0.05 were considered statistically significant.

RESULTS

Effects of prophylactic IVIG on development of the acute EAE attack

In the saline control group the animals resumed normal growth curves a few days after the inoculation and infusion procedures. The animals in this group did not develop any sign of disease. The rats injected with the encephalitogenic emulsion receiving no further treatment (EAE controls and EAE+placebo group) developed an acute EAE attack at days 6-7 p.i. The initial neurological symptoms included loss of tail tone and were accompanied by a reduction in body weight (Figures 1 and 2). The observed reduction in body weight was aggravated until the acute EAE attack reached its maximum at days 10-11 p.i. At this time point, the EAE symptoms also included paralysis of the tail and paresis or paralysis of the hind limbs. Intravenous administration of 1 g/kg IVIG at days O and 1 p.i. significantly reduced the loss of body weight during the acute attack (p

Effects of prophylactic versus therapeutic IVIG treatment on long-term EAE

Infusions of IVIG were administered either prophylactically at the time of EAE induction at days 0 and 1 p.i. or therapeutically at the beginning of the acute EAE attack at days 8 and 9 p.i. After immunization, the animals were monitored for 28 days. The symptoms of EAE were observed 1 week after the immunization, where the rats in the placebo group had a profound weight loss. If the animals received the IVIG at days 0-1 p.i., the loss of body weight was significantly reduced (Figure 3A). When IVIC was administered therapeutical Iy at days 8-9 p.i., when the symptoms of EAE were evident, the treatment had no effect on the body weight (Figure 3B). The development of the neurological EAE symptoms coincided with the body weight changes, and prophylactic treatment with immunoglobulin resulted in a less severe course of the disease with only mild symptoms of EAE (Figure 4A). When the infusions of IVIG were given therapeutically during established EAE, no significant treatment effects could be observed (Figure 4B). The EAE incidence after prophylactic IVIG treatment was 75 (9/12) versus 100% (12/12) in animals receiving therapeutic IVIG infusions at days 8-9 p.i.

Histological findings after IVIG treatment

Tissue samples from the CNS were obtained at day 11 p.i. during the acute EAE attack and at day 28 p.i. The principal histopathological findings were a pronounced inflammatory response in both the brain and spinal cord, predominantly as perivascular infiltrations, but demyelination was also observed (Figure 5). Prophylactic administration of IVIG at days 0-1 significantly inhibited the inflammatory response as evaluated by the blinded scoring of the histological findings (Figure 6). The effects of prophylactic IVIG treatment were evident during the acute attack at day 11 p.i. (IVIG average score 1.1±0.2 versus placebo 1.8±0.2, P=0.040) and also at the later stages of EAE at day 28 p.i. (IVIG 1.0±0.1 versus placebo 1.6±0.2, P=0.029). The therapeutic administration of IVIG at days 8-9 p.i. did not affect inflammation or demyelination in the CNS (IVIG 1.6±0.3 versus placebo 1.5±0.2). All the tissue samples from animals immunized with encephalitogenic emulsion exhibited varying degrees of inflammation, whereas all the rats in the saline control group received the inflammation score 0.

Correlation of EAE symptoms and histological findings

Correlation was calculated between the body weight, clinical score at the last day of experiments, and the histopathological scoring. In this study, 86 animals were included of which 76 survived until the end of the experiment at days 11 or 28 p.i.. EAE scores and inflammation scores were positively correlated with a correlation coefficient (r) of 0.72 (P

DISCUSSION

In the present study we found significant effects of IVIG on the acute EAE attack and the long-term disease course. Eoss of body weight and neurological EAE symptoms were only inhibited when IVIG treatment was administered prophylactically at the time of immunization. The therapeutic infusions of IVIG during established EAE did not ameliorate the EAE symptoms and had no beneficial effects on body weight. These observations support previous findings by others24,25. Moreover, we studied the inflammatory response in the brain and spinal cord by histopathology at days 11 and 28 after the induction of EAE. The development of EAE was associated with extensive inflammation in the CNS, and areas of demyelination were also observed. Infusions of IVIG significantly inhibited the histopathology, but only when administered prophylactically. Considering the short duration of treatment applied in this study (days 0 and 1 p.i.), the observations demonstrate that high-dose IVIG treatment is a very effective anti-inflammatory remedy with substantial effects on the CNS lesion formation in EAE. The results also indicate that IVIG acts during the early phase of the immune response in EAE, as no improvement was observed when the infusions were given at days 8-9 p.i. when the symptoms of EAE were evident.

The exact mechanism by which IVIG influences the immune response in CNS inflammation is not known. Considering the many anti-inflammatory and immunomodulatory effects of the polyclonal IVIG preparations, it is likely that several mechanisms act in concert to prevent or ameliorate the development of EAE symptoms. IVIG may be of benefit in autoimmune diseases due to the suppression of harmful autoantibodies through anti-idiotypic interactions12,26, but may also be effective in autoimmune diseases primarily mediated by pathogenic T lymphocytes through inhibitory effects on these cells27,28. The observed efficiency of prophylactic IVIG infusions in EAE is in accordance with the ability of IVIG to inhibit the activation of T cells, which are pivotal in establishing EAE. However, the later stages of the autoimmune T-cell response in EAE are possibly inhibited by the IVIG treatment as well, as it has been shown that EAE by adoptive transfer is inhibited only by IVIG pre-treatment of the activated, encephalitogenic T cells in vitro, not by in vivo IVIG treatment25. In addition to the inhibitory effects on antibodies and lymphocyte function, IVIG may also suppress injury mediated by the complement system15, interfere with the cytokine network29, and affect the function of phagocytes13. These effects of IVIG may contribute to the reduced inflammatory response and demyelination in the CNS, as all of these cellular and humoral immune reactions participate in the development of the EAE disorder20,21. In accordance with the classification of demyelinating CNS lesions described by Lassmann and co-workers2, IVIG may be particularly effective in the type II lesions characterized by the deposition of complement components and antibodies.

Although the modes of actions mentioned above are anti-inflammatory, IVIG treatment may be a suitable strategy in suppressing neurodegeneration. In MS, the pathophysiological sequence of events is thought to involve a primary inflammatory process with cellular infiltration of the CNS followed by demyelination that allows subsequent transection of demyelinated axons in the affected regions1. Apparently, axonal damage is responsible for the irreversible neurological deficits in MS patients and seems to be prevented by protective remyelination in shadow plaques22. In the Theilers murine encephalomyelitis virus (TMEV) model of immune-mediated demyelination it has been shown that immunoglobulin has the potential to act through myelin repair mechanisms30,31. However, the possible remyelinating effects of IVIG in MS have not been confirmed in clinical trials that were designed to evaluate the persistence of stable deficits after optic neuritis or permanent motor deficits in MS32,33. In a recent trial, MS patients receiving IVIG in combination with i.v. methylprednisolone did not recover faster or more completely from acute relapses than patients treated with methylprednisolone alone, and the trial could not corroborate the hypothesis that IVIG enhances remyelination in the acute MS plaque34. IVIG treatment of relapsing-remitting MS has been studied in several controlled trials that demonstrated a significant reduction in the annual relapse rate, a decreased disability score (EDSS), and a reduction in the number of new lesions by MRI after IVIG treatment8,9,35,36. A meta-analysis of four randomized, placebo-controlled studies was recently published confirming that patients with the relapsing type of MS benefit significantly from IVIG treatment10. On the other hand, clinical trials using IVIG infusions for treatment of secondary progressive MS have proven negative37.

The existing experimental and clinical data may seem conflicting as the beneficial effect of IVIG on the relapse rate in MS is high, the magnitude being comparable with that of the first-line compounds interferon-β and glatiramer acetate. The development of EAE symptoms in the experimental model may, however, be considered parallel to a single relapse in the relapsing-remitting type of MS. Therefore, the advantageous effects of IVIG may be regarded as the result of an ability to prevent the development of relapses through various immunomodulatory mechanisms.

To summarize, we observed that IVIG protected against actively induced EAE, and the treatment effect was associated with a significant suppression of inflammatory lesions within the CNS. The data suggest that IVIG acts primarily through the early phase of the immune response, as the therapeutic administration of IVIG had no effect on the course of EAE. It remains to be clarified, which of the numerous anti-inflammatory effects of IVIG are of special importance in CNS inflammation and demyelination.

ACKNOWLEDGEMENTS

The authors acknowledge the technical assistance of Inge Møller, Neurobiology Research Unit, Copenhagen University Hospital, Denmark, Ann Meisler, Jan Lauritzen, and Bodil Sneskov, Laboratory of Neuropathology, Copenhagen University Hospital, Denmark. Parts of this study were presented at the Charcot Symposium 2003, Lisbon, Portugal. Bayer AC, Leverkusen, Germany, provided the intravenous immunoglobulin used in this study. This study was supported by funds from The H0rslev Foundation, Danielsens Foundation, The Danish MS Society, The Dir. Ejnar Johnsen Foundation, Warwara Larsen Foundation, The Augustinus Foundation, Novo Nordisk Foundation, Sigurd and Addie Abrahamson's Legacy, and Karen A. Tolstrups Foundation.

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Signe Humle Jorgensen*, Poul Erik Hyldgaard Jensen*, Henning Laursen[dagger] and Per Soelberg Sorensen*

* Danish MS Centre, Copenhagen University Hospital, Rigshospitalet sect. 9202, DK-2100 Copenhagen, Denmark

[dagger] Laboratory of Neuropathology, Copenhagen University Hospital, Rigshospitalet, DK-2100 Copenhagen, Denmark

Correspondence and reprint requests to: Signe Humle Jorgensen, MS Research Unit, Copenhagen MS Centre, Copenhagen University Hospital, Rigshospitalet sect. 9202, DK-2100 Copenhagen, Denmark. [shumle@nru.dk] Accepted for publication April 2005.

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