Effect of dimethyl sulfoxide on blood-to-brain transfer of alpha-aminoisobutyric acid: examination of regional blood-brain barrier function

The effect of dimethyl sulfoxide (DMSO) on brain capillary permeability has been controversial. We have studied the effect of DMSO on unidirectional transport of alpha-aminoisobutyric acid (AIB) across the blood-brain barrier (BBB) in rats. Rats were treated with 15% DMSO intraperitoneally (i.p.), intravenously (i.v.) or by an i.p. injection in combination with an i.v. injection, or in some cases intra-arterially by rapid infusion into left external carotid artery. The unidirectional blood-to-brain transfer constant (Ki) for AIB was measured in each group after the animals were killed. DMSO administration did not significantly increase Ki as compared to control Kj. These results show that it is unlikely that DMSO increases the permeability of BBB and therefore do not support the proposal that DMSO can act as a carrier at the BBB for compounds with restricted vascular permeability.     

von-Willebrand factor influences blood brain barrier permeability and brain inflammation in experimental allergic encephalomyelitis

Weibel-Palade bodies within endothelial cells are secretory granules known to release von Willebrand Factor (VWF), P-selectin, chemokines, and other stored molecules following histamine exposure. Mice with a disrupted VWF gene (VWFKO) have endothelial cells that are deficient in Weibel-Palade bodies. These mice were used to evaluate the role of VWF and/or Weibel-Palade bodies in Bordetella pertussis toxin-induced hypersensitivity to histamine, a subphenotype of experimental allergic encephalomyelitis, the principal autoimmune model of multiple sclerosis. No significant differences in susceptibility to histamine between wild-type and VWFKO mice were detected after 3 days; however, histamine sensitivity persisted significantly longer in VWFKO mice. Correspondingly, encephalomyelitis onset was earlier, disease was more severe, and blood brain barrier (BBB) permeability was significantly increased in VWFKO mice, as compared with wild-type mice. Moreover, inflammation was selectively increased in the brains, but not spinal cords, of VWFKO mice as compared with wild-type mice. Early increases in BBB permeability in VWFKO mice were not due to increased encephalitogenic T-cell activity since BBB permeability did not differ in adjuvant-treated VWFKO mice as compared with littermates immunized with encephalitogenic peptide plus adjuvant. Taken together, these data indicate that VWF and/or Weibel-Palade bodies negatively regulate BBB permeability changes and autoimmune inflammatory lesion formation within the brain elicited by peripheral inflammatory stimuli.     

Early detection of liposome brain localization in rat experimental allergic encephalomyelitis

Blood-brain barrier (BBB) permeability increases prior to the development of clinical signs in early-stage multiple sclerosis (MS). Detection of subtle changes would thus be helpful for diagnostic purposes and rapid therapeutic decisions before new episodes. Since multiple sclerosis and experimental allergic encephalomyelitis (EAE) have numerous common features, in particular BBB-permeability characteristics, and since we have previously shown that BBB localization is disturbed by tumors, embolism, and mannitol injection, we investigated BBB-liposome permeability in an EAE rat model. Twenty young male Lewis rats received a single intradermal inoculation of guinea-pig spinal cord. The effect of the Freund's adjuvant and spinal cord alone on brain permeability were also assessed. In order to compare solution permeability and liposome localization, radioactive liposomes and, 1 h later, 99mTc-DTPA were injected intravenously. Scintigraphic acquisitions were obtained to follow the biodistribution of radioactivity in the whole body. Each rat was subjected to a first examination before inoculation and then every two days until completion and may be considered as its own control. EAE induced a previously unreported increase in global-body permeability, probably due to inflammation. Liposome brain localization and brain/heart ratio were significantly different between normal animals and those with early-stage EAE (before appearance of clinical signs) and distinguished between different disease stages in clinically patent EAE. The index of disease progression was modified earlier than with 99mTc-DTPA injection. One explanation may be particle pick-up by circulating macrophages, which cross the BBB during this pathology. For clinical applications, experiments must be confirmed on models more reliable for human multiple sclerosis.     

Hydroxyzine inhibits experimental allergic encephalomyelitis (EAE) and associated brain mast cell activation

Experimental allergic encephalomyelitis (EAE) has been used as an animal model for the human demyelinating disease multiple sclerosis (MS). In acute MS or EAE, early disruption in the integrity of the blood-brain-barrier (BBB) precedes brain infiltration by inflammatory cells or any clinical evidence of disease. BBB permeability could be affected by vasoactive mediators and cytokines released from perivascular brain mast cells. We investigated the number and degree of activation of brain mast cells in EAE and the effect of the heterocyclic histamine-1 receptor antagonist hydroxyzine, a piperazine compound known to also block mast cells. Acute EAE was induced in Lewis rats by immunization with whole guinea pig spinal cord homogenate and complete Freund's adjuvant (CFA). A second group of animals were treated orally with hydroxyzine for one day before immunization and then continuously for 14 days. Control rats were treated with CFA or hydroxyzine alone. The clinical progression of EAE was assessed on days 10, 12 and 14 after immunization. The number of metachromatic mast cells and the degree of degranulation was assessed in the thalamus with light microscopy. At day 14, there was a three-fold increase in the number of brain mast cells with EAE, as compared to controls. These cells were positive for the immunoglobulin E binding protein (FcepsilonRI), while those from control rats were not. Over 40% of all thalamic mast cells studied in EAE showed partial staining or extruded secretory granule indicative of secretion. Hydroxyzine treatment inhibited (p<0.05) the progression and severity of EAE by 50% and the extent of mast cell degranulation by 70% (p<0.05). These findings indicate that brain mast cells are associated with EAE development and that inhibition of their activation correlates positively with the clinical outcome.     

Investigation of the blast transformation reaction of lymphocytes in guinea pigs with experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) was induced in guinea pigs by injection of myelin from homologous brain in Freund's complete adjuvant. In the incubation period of EAE some animals showed a marked increase in the spontaneous proliferative activity of the blood lymphocytes, assessed in cultures using thymidine-H³. With the development of clinical manifestations of the disease, the spontaneous activity of the lymphocytes reached a maximum and was exhibited in the overwhelming majority of animals. When the clinical picture of EAE was well marked, the response of the lymphocytes to stimulation by myelin preparations was observed mainly if the spontaneous mitotic activity of the cells was low or only moderately increased. Specific antigenic stimulation in vivo connected with the immunopathological process could be the cause of the increasing spontaneous activity of the lymphocytes. The state of nonspecific reactivity of the lymphocytes to phytohemagglutinin in EAE was indistinguishable from that in the control animals.     

Effects of dimethyl sulfoxide and colchicine on the resorption of experimental amyloid

Summary The induction of amyloid in C₃H mice by either casein solution or complete Freund's adjuvant emulsion with Mycobacterium butyricum was confirmed by partial splenectomy. The animals were autopsied after treatment w ith dimethyl sulfoxide (550 mg/kg, 50 times), colchicine (0.02 mg/kg, 15–37 times), or saline solution as a control. Detailed histological comparisons of biopsy and autopsy spleens provided evidence that dimethyl sulfoxide was significantly effective in the resorption of amyloid, while in the animals treated with colchicine amyloid deposition was increased. The effect of dimethyl sulfoxide was discussed with reference to the modification of amyloid fibrils.Supported by a Research Grant for Specific Diseases from the Ministry of Health and Welfare and the Grant-in-Aid for Scientific Research of the Ministry of Education.     

Relationship between the incidence of experimental allergic encephalomyelitis and the development of adjuvant disease

Experimental allergic encephalomyelitis (EAE) was induced with equal incidence in groups of rats regardless of whether the route of brain vaccine injection was intracutaneous and into hind foot pads or intraperitoneal. Rats which were injected intracutaneously and into hind foot pads either developed EAE within 10–16 days or showed signs of what we have termed `adjuvant disease'. Intraperitoneally injected animals that failed to develop demyelinating disease showed almost diffuse proliferative peritonitis, but those with EAE had a more circumscribed granulomatous proliferation in the upper part of abdomen. Suppression of EAE was obtained when the oily suspension of mycobacteria without brain was injected intraperitoneally within 11 days after the inoculation of brain in adjuvant mixture intradermally and into foot pads. No suppression of EAE was seen when adjuvant alone was injected intraperitoneally 12 or 14 days after brain in adjuvant mixture. The results are discussed in terms of a hypothesis based on the competition of antigens.     

Vascular permeability changes in the central nervous system of rats with hyperacute experimental allergic encephalomyelitis induced with the aid of a substance from Bordetella pertussis.

Development of hyperacute experimental allergic encephalomyelitis in Lewis rats after intraperitoneal administration of a mixture of guinea pig spinal cord emulsion and pertussigen from Bordetella pertussis was accompanied by an increase in vascular permeability in the central nervous system. The increased permeability was most striking in the spinal cord and seemed to be associated with the ascending development of paralysis. Rats that had completely recovered from paralysis did not have any increased permeability in the central nervous system. Rats which developed paralysis after inoculation with either guinea pig spinal cord emulsion alone or with complete Freund adjuvant had only a small degree, if any, of increased permeability in the vascular system of the central nervous system.     

Passive transfer of experimental allergic encephalomyelitis with lymphocytes collected from brains of immunized guinea pigs

Lymphocytes were collected from the brains of guinea pigs immunized with syngeneic spinal cord emulsified in complete Freund's adjuvant. Sixteen to 32 X 10(6) lymphocytes were inoculated into the vein of each inbred guinea pig (NIH 13). Recipient animals without clinical signs of experimental allergic encephalomyelitis (EAE) were sacrificed 20 days later. Some of the recipients had perivascular infiltrates of a large number of mononuclear cells mostly in choroid plexus and meninges of the brain and spinal cord. Demyelination, which was not so intense, was also observed in the vicinity of the perivascular infiltrates. Thus, the lymphocytes from the brain as well as lymph node and spleen were clear to have the ability to transfer EAE.     

An investigation into the possible involvement of platelet activating factor in experimental allergic encephalomyelitis in rats

Rats developing experimental allergic encephalomyelitis have been treated with intra-venous injections of Platelet-Activating Factor on days 5,6 and 7 post adjuvant injection. Other rats have been treated with one of two different PAF antagonists or vehicle. The animals injected with PAF on day 5 post adjuvant developed a more severe form of the disease at an earlier time point than did control animals. Animals treated with the PAF antagonists did not develop the disease to any great extent. Treatment with the vehicle had no effect compared to control. These results implicate PAF in the aetiology of this model of multiple sclerosis and may suggest a role for PAF antagonists in the treatment of this disease.     

Mechanisms in the pathogenesis of post-infectious vaccinia virus encephalomyelitis in the mouse.

After intracranial replication of a neurotropic strain of vaccinia in mouse brain, analysis of the purified virus preparation reveals the presence of at least one host protein on the virus which was identified as the myelin basic protein. Vaccinia virus Elstree, a dermotropic virus may substitute for complete Freund's adjuvant (CFA) in inducing experimental allergic encephalomyelitis (EAE). Guinea pigs challenged with virus-myelin emulsions without CFA developed clinical and histological signs of EAE.     

PASSIVE TRANSFER OF EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS WITH LYMPHOCYTES COLLECTED FROM BRAINS OF IMMUNIZED GUINEA PIGS

Lymphocytes were collected from the brains of guinea pigs immunized with syngeneic spinal cord emulsified in complete Freund's adjuvant. Sixteen to 32 times 10⁶ lymphocytes were inoculated into the vein of each inbred guinea pig (NIH 13). Recipient animals without clinical signs of experimental allergic encephalomyelitis (EAE) were sacrificed 20 days later. Some of the recipients had perivascular infiltrates of a large number of mononuclear cells mostly in choroid plexus and meninges of the brain and spinal cord. Demyelination, which was not so intense, was also observed in the vicinity of the perivascular infiltrates. Thus, the lymphocytes from the brain as well as lymph node and spleen were clear to have the ability to transfer EAE.     

Unresponsiveness to experimental allergic encephalomyelitis in Lewis rats pretreated with complete Freund's adjuvant

A single injection of complete Freund's adjuvant blended with aluminum hydroxide gel (ALU-CFA) was successfully used to prevent clinical as well as histologic experimental allergic encephalomyelitis (EAE) in Lewis rats for more than 330 days. Adjuvant preparations with Mycobacterium tuberculosis were more potent than those with Mycobacterium butyricum. Neither the removal of the ALU-CFA inoculum nor a splenectomy 1 month after immunization arrested the adjuvant induced unresponsiveness. However cyclophosphamide restored responsiveness in more than half of the treated animals when applied 2 days before the encephalitogenic challenge at a dose of 20 mg/kg. Passive EAE was not prevented by the ALU-CFA pretreatment. The disease was induced by the transfer of 4 X 10(6) T lymphocytes of a cell line reactive against myelin basic protein. This indicates that the adjuvant prevents EAE at the inductive rather than at the effector phase of the autoimmune response.     

Protection against experimental allergic encephalomyelitis with complete Freund's adjuvant is unaffected by prostaglandin synthesis inhibition

Pretreatment with complete Freund's adjuvant blended with aluminium hydroxide (ALU-CFA) prevents the clinical as well as histological manifestation of experimental allergic encephalomyelitis (EAE) in Lewis rats. Suppression of prostaglandin biosynthesis with the cyclooxygenase inhibitor piroxicam (10 mg/kg body weight) from day 2 before to day 17 after EAE induction could not restore responsiveness in pretreated animals. In contrast piroxicam increased ALU-CFA-induced suppression of autoantibodies against myelin basic protein. In controls not pretreated with ALU-CFA, clinical signs of EAE were attenuated by piroxicam, whereas mononuclear infiltration of the brain remained unchanged. Therefore it is unlikely that prostaglandins exert an important function in the regulation of immune responses in this model of autoimmunity.     

A new pattern of spinal-cord demyelination in guinea pigs with acute experimental allergic encephalomyelitis mimicking multiple sclerosis.

A technique is described for producing large demyelinating lesions of the spinal cord in the guinea pig. Guinea pigs were pretreated by immunization with ovalbumin and water-soluble adjuvant (N-acetyl-muramyl L-alanyl D-isoglutamine, MDP) in water-in-oil emulsion (Freund''s incomplete adjuvant). They were given a large dose (10 mg) of ovalbumin i.p. one month later. After a few weeks the animals were sensitized with guinea-pig basic protein in Freund''s complete adjuvant. Five out of 11 animals developed large, distinctive, sharply demarcated, symmetrical demyelinating lesions within 30 days. These lesions occurred in the dorsal and anterior columns, root entry zones and subpial region of the spinal cord. Histology showed a considerable amount of free lipids. There were also infiltrative lesions of classical experimental allergic encephalomyelitis (EAE) of normal severity in the same animals. The demyelinating lesions resembled those seen in multiple sclerosis in their location and extent in the spinal cord and in the presence of free lipids. Control experiments indicated that pretreatment with ovalbumin/MDP and the second injection of ovalbumin was necessary for all the demyelination; moreover guinea pigs immunized with basic protein in Freund''s complete adjuvant or Freund''s incomplete adjuvant plus MDP without pretreatment only developed classical EAE with minimal or no demyelination.     

Experimental allergic encephalomyelitis.-- The effect of dexamethasone on growth and proliferation in the draining lymph node.

Dexamethasone, when administered from day 0 to day 9 following the sensitizing injection of encephalitogenic protein in complete Freund's adjuvant, suppressed both the characteristic enlargement of the paracortical area of the draining lymph node and the development of clinical and histopathological signs of experimental allergic encephalomyelitis. Division of cells within the node was not inhibited and therefore the effect of dexamethasone appeared to be a consequence of reduced migration of lymphocytes into the node.     

Tumour necrosis factor-alpha causes an increase in blood-brain barrier permeability during sepsis

Blood-brain barrier (BBB) permeability during sepsis with Escherichia coli or Streptococcus pneumoniae was examined in a mouse model and measured by a circulating beta-galactosidase tracer. The leakage of brain microvascular vessels during sepsis was confirmed by transmission electron microscopic examination of brain tissues stained with horseradish peroxidase. The increase of BBB permeability induced by E. coli and S. pneumoniae, which was maximal at 3 h and 12 h after injection, respectively, was transient because of rapid clearance of the bacteria from the blood. Tumour necrosis factor-alpha (TNF-alpha) was stained on microvascular vessels of the brain during sepsis and intravenous injection of recombinant TNF-alpha also increased the BBB permeability. The increase in BBB permeability induced by either E. coli or S. pneumoniae could be inhibited by anti-TNF-alpha antibody. It was concluded that circulating TNF-alpha generated during sepsis induced the increase in BBB permeability.     

Dissection of adjuvant and suppressive effects of mycobacteria in experimental allergic encephalomyelitis production

Dark August rats exhibit clinically and histologically verified experimental allergic encephalomyelitis (EAE) when immunized with appropriate antigen (nervous tissue, myelin basic protein) emulsified in complete Freund's adjuvant (CFA). We provide evidence that 6,6'-trechalose dymicolate (TDM) incorporated in incomplete Freund's adjuvant replaces CFA in EAE induction. The animals that recovered from EAE were resistant to the reinduction of the disease irrespectively whether Mycobacterium tuberculosis or TDM was used as an adjuvant. Finally, pretreatment with CFA alone was sufficient for prevention of disease elicited by challenge with encephalitogen + CFA. However, TDM, despite its adjuvant capacity when applied prior to the induction of the disease with encephalitogen + CFA, did not exhibit any protective effect. Thus, our study implicates that adjuvant and suppressive capacities of M. tuberculosis may be related to the different determinants of the microorganisms, TDM possessing the adjuvanticity only.     

Gangliosides improve the outcome of experimental allergic neuritis (EAN)

The effect of gangliosides on the clinical expression of experimental allergic neuritis (EAN), a PNS counterpart of experimental allergic encephalomyelitis (EAE), was tested in Lewis rats sensitized with bovine intradural root myelin in complete Freund's adjuvant (CFA). A mixture of bovine brain gangliosides (GM1, GD1a, GD1b, GT1b) was injected intramuscularly at a daily dose of 20 mg per kg beginning 6 days post inoculation. The results show that GA treatment considerably reduces the incidence of mortality, delays its occurrence, and reduces the severity of clinical scores when either the whole population or survivors only are considered. Benefit from GA treatment appeared roughly proportional to disease severity. The neuronotrophic and anti-inflammatory effects possibly play a role in GA protecting effect.     

Inhibition of blood-brain barrier disruption in experimental allergic encephalomyelitis by short-term therapy with dexamethasone or cyclosporin A

Double radioisotope measurement of neurovascular integrity in Lewis rats inoculated for experimental allergic encephalomyelitis (EAE) showed abnormal elevation of albumin extravasation in the cerebellum, medulla-pons and cervical spinal cord at the time of clinical manifestation. Therapeutically administered dexamethasone (Dex) (0.1–1 mg/kg body weight) or cyclosporin A (CsA) (25–75 mg/kg body weight) dosedependently reduced albumin movement across the blood-brain barrier (BBB). Dex at a dose of 1 mg/kg completely suppressed abnormal BBB permeability in all tissues (P≤0.001), while CsA at the highest dose of 75 mg/kg achieved highly significant (P≤0.001), but not complete, suppression of aberrant barrier leakage in the areas studied. The implications of these findings to possible drug action at the immunocompromised cerebrovasculature are discussed.