Antigen-dependent intrathecal antibody synthesis in the normal rat brain: tissue entry and local retention of antigen-specific B cells.

The intrathecal Ab response to Ag introduced into the normal brain has not been fully explored. Involvement of Ag-specific, peripheral B cells in an intrathecal response was studied using a normal rat model of Ag infusion through an indwelling cannula into defined brain sites, while maintaining a functionally intact blood-brain barrier. Specific Ab was detected in serum and cerebrospinal fluid. The intrathecal response is first detectable at day 14. Isoelectric focusing of cerebrospinal fluid reveals banding patterns consistent with local Ab production. To increase Ag-specific, circulating peripheral lymphocytes available for trafficking to Ag-stimulated brain and for enhancing intrathecal Ab synthesis, rats were preimmunized peripherally. Subsequently, Ag or saline (control) was infused through the cannula. Under this protocol, intrathecal synthesis is detectable earlier (day 5 postinfusion). Immunohistochemical studies at the infusion site assessed Ag-specific B cells, T cells, and activated APCs. Rats receiving peripheral preimmunization followed by Ag into caudate nucleus have far greater numbers of these cells, including plasma cells, within the infusion site compared with saline controls. Results confirm previous indirect evidence of intrathecal Ab synthesis in normal rat brain and provide the first direct evidence for B cell trafficking across normal brain barriers plus retention at the Ag deposition site. Our studies indicate that the normal brain microenvironment supports development of Ag-directed humoral immunity. We propose that immune privilege in normal brain is characterized by down-regulation of cell-mediated but not Ab immune responses within the central nervous system.

Growth of P511 mastocytoma cells in BALB/c mouse brain elicits CTL response without tumor elimination: a new tumor model for regional central nervous system immunity.

We have developed a murine model to explore the tumor-specific CTL response in the immune-privileged central nervous system using P511 mastocytoma cells. Three strains with varying degrees of histocompatibility to P511 cells (CD-1, allogeneic; BALB/c, minor histoincompatible; DBA/2, syngeneic) received tumor cells (10(4)) into the putamen 7 days after cannula implantation, when the blood-brain barrier was functionally intact. Without exception, tumor formed reproducibly by day 7 in all strains. Tumor rejection occurred in CD-1 but not in BALB/c and DBA/2 mice. Using a flank injection site, both CD-1 and BALB/c, but not DBA/2 mice, ultimately rejected flank tumors. Analysis of tumor-specific CTL in BALB/c spleens revealed that P511 administration into brain or flank elicited similar responses: no fully activated CTL were detectable but a significantly expanded population of nonkilling precursors of CTL (pCTL) were present. A P511 cell-specific pCTL population was also identified at the brain tumor site 14 days post-tumor introduction, indicating that pCTL, generated in the periphery, traffic to the tumor site in brain. These data indicate that failure to reject tumor in brain is neither due to lack of afferent stimulation nor to inability of peripheral effectors (P511 cell-specific pCTL) to reach the tumor site. We hypothesize that these effector cells are prevented from developing into fully activated CTL by conditions within the central nervous system microenvironment that down-regulate CTL development.

Ontogeny of blood-brain barrier function in ovine fetuses, lambs, and adults.

The ontogeny of regional blood-brain barrier function was quantified with the rate constant for influx (Ki) across the blood-brain barrier with the small molecular weight synthetic, inert hydrophilic amino acid alpha-aminoisobutyric acid (AIB) in chronically instrumented early (87 days of gestation, 60% of gestation) and late (137 days of gestation, 90% of gestation) gestation fetal, newborn (3 days of age), older (24 days of age), and adult (3 years of age) sheep. The Ki was significantly (P < 0.05) lower in the brain regions of the adult sheep and in most brain regions of newborn and older lambs compared with fetuses at 60 and 90% of gestation. The Ki exhibited regional brain heterogeneity (P < 0.05) in the five groups. The patterns of regional heterogeneity were accentuated (P < 0.05) in the younger groups. We conclude that ontogenic decreases in blood-brain barrier permeability are observed in ovine fetuses from 60% of gestation to maturity in the adult.

Elevated cerebrospinal fluid IgA in humans and rats is not associated with secretory component.

Immunoglobulin A (IgA) is transported across mucosal tissue membranes covalently bound to secretory component (SC). To determine if this receptor-mediated process also occurs at central nervous system (CNS) boundaries, cerebrospinal fluid (CSF) and serum from patients with CNS neuroinflammatory disease were analyzed for IgA and SC. Excess CSF IgA was detected in six of 24 patients, but no significant CSF SC was detected. In a parallel study using a rat model with normal brain barriers, inactivated lymphocytic choriomeningitis virus was microinfused into CSF. Elevated CSF IgA was detected in four of six rats, yet the proportion of secretory IgA was again insignificant compared to normal exocrine fluids (bile, semen). There does not appear to be a secretory IgA immune system at CNS boundaries and elevated CSF IgA is attributed to intrathecal synthesis.

Effects of arginine vasopressin and atriopeptin on glial cell volume measured as 3-MG space.

This study evaluates the hypothesis that arginine vasopressin (AVP) and atriopeptin, peptide hormones synthesized and released within the brain, are regulators of brain cell volume using cultured astroglial cells derived from newborn rats. Cell water content, regarded as volume, was measured in defined, serum-free medium as the 3-O-methylglucose (3-MG) space. Initial experiments established conditions such that glucose, which competes with 3-MG for the glucose carrier, would not interfere with the measurement of the 3-MG space. AVP increased the 3-MG space of glial cells by an average of 25% between 30 and 120 min of exposure, whereas atriopeptin decreased it by 32%. The 3-MG space remained close to normal after coadministration of both peptides. The AVP-dependent increase in 3-MG space was blocked both by the V1 antagonist d(CH2)5Tyr(Me)AVP (Manning compound) and by the cotransport inhibitor, bumetanide. Results are consistent with a role for AVP and atriopeptin in the homeostasis of atroglial cell volume.

Cervical lymphatics, the blood-brain barrier and the immunoreactivity of the brain: a new view.

This new view of the immunoreactivity of the normal brain is based on three key components. First, there is an active and highly-regulated communication between the brain and the central immune organs. Secondly, the connection from the brain to the draining nodes is much larger than previously appreciated. And third, the blood-brain barrier, by virtue of its selective permeability properties, contributes to the regulation of immunoregulatory cells and molecules in the brain cell microenvironment.

Afferent and efferent arms of the humoral immune response to CSF-administered albumins in a rat model with normal blood-brain barrier permeability.

Cerebrospinal fluid (CSF) and serum antibody responses to albumin administered into CSF or muscle have been compared with respect to titer, isotype profile and complement-fixing activity in a rat model with normal brain barrier function. CSF/serum titer ratios and the ratio of IgG subclasses, IgG1/IgG2, were both elevated following CSF immunization. In contrast, there was no difference in complement-fixing activity between antibodies elicited by the two routes of immunization. It is suggested that intrathecal antibody synthesis accounts for the elevated CSF antibody titers in CSF-immunized rats, providing the first example of central nervous system antibody synthesis in an animal with normal brain barrier permeability.

Drainage of brain extracellular fluid into blood and deep cervical lymph and its immunological significance.

Cerebral extracellular fluids drain from brain to blood across the arachnoid villi and to lymph along certain cranial nerves (primarily olfactory) and spinal nerve root ganglia. Quantification of the connection to lymph in rabbit, cat and sheep, using radiolabelled albumin as a marker of flow, indicates that a minimum of 14 to 47% of protein injected into different regions of brain or cerebrospinal fluid passes through lymph. The magnitude of the outflow to lymph is at variance with the general assumption that the absence of conventional lymphatics from the brain interrupts the afferent arm of the immune response to brain antigens. The immune response to antigens (albumin or myelin basic protein) introduced into the central nervous system (CNS) has been analysed using a rat model with normal brain barrier permeability. The micro-injection of antigen into brain or cerebrospinal fluid elicits a humoral immune response, with antibody production in cervical lymph nodes and spleen, and also affects cell-mediated immunity. Furthermore, antigen may be more immunogenic when administered into the CNS than into conventional extracerebral sites. Clearly, the afferent arm of the immune response to antigens, within the CNS, is intact. Modern studies suggest that the efferent arm is also intact with passage of activated lymphocytes into the brain. Results support a new view of CNS immunology which incorporates continuous and highly regulated communication between the brain and the immune system in both health and disease.

Ovalbumin is more immunogenic when introduced into brain or cerebrospinal fluid than into extracerebral sites.

The magnitudes of serum antibody responses to ovalbumin have been compared following immunization via cerebral or extracerebral sites in Sprague-Dawley rats. In central nervous system (CNS)-immunized rats, conditions were designed to ensure normal brain barrier permeability. Extracerebral immunization was via the footpad or along pathways of antigen outflow from the CNS. The relative immunogenicity of different injection sites is: CSF greater than brain tissue greater than extracerebral sites. Enhancement of the antibody response to CNS-administered antigen appears to depend on events initiated within the CNS, since ovalbumin injected into blood (which reaches the spleen) or nasal submucosa (which drains to cervical nodes) fails to elicit a similar response.

Blood-brain barrier integrity and brain water and electrolytes during hypoxia/hypercapnia and hypotension in newborn piglets.

This study examines the effects of hypoxia/hypercapnia and hypoxia/hypercapnia with hypotension (hypotensive-hypoxia/hypercapnia) on blood-to-brain transfer constants (K1) for sodium and mannitol and brain water and electrolyte contents in newborn piglets. Hypoxia/hypercapnia was induced for 60 min with the piglets breathing a gas mixture of 15% carbon dioxide, 10-12% oxygen, and 73-75% nitrogen adjusted to achieve an arterial pH less than 7.15, pO2 less than 40, and pCo2 greater than 60 mmHg and hypotension for 20 min by rapid phlebotomy to achieve a mean arterial blood pressure less than 40 mmHg. Piglets were studied during 1 h of, and 24 h after resuscitation from hypoxia/hypercapnia (arterial pH 6.9 +/- 0.18, pO2 36 +/- 6 mmHg, pCO2 68 +/- 8 mmHg, mean +/- S.D.) and 10 min, and 24 h after resuscitation from hypotensive-hypoxia/hypercapnia (mean arterial blood pressure 28 +/- 10 mmHg, mean +/- S.D.). Values for K1 for sodium and mannitol, measured using the integral technique were 15.9 and 5.2 ml.g-1.min-1 x 10(4) respectively, in 2-4-day-old controls, suggesting that the barrier is fully developed in newborn piglets. Values were not different during or after hypoxia/hypercapnia or 24 h after hypotensive-hypoxia/hypercapnia. Ten to forty min after hypotensive-hypoxia/hypercapnia, there was a proportional decrease in the K1 for sodium and mannitol of about 40%. These results suggest that the newborn piglet is similar to the adult with respect to impermeability of the blood-brain barrier to ions and small molecules and resistance of this barrier to systemic hypoxia/hypercapnia and hypotension.(ABSTRACT TRUNCATED AT 250 WORDS)

Myelin basic protein infused into cerebrospinal fluid suppresses experimental autoimmune encephalomyelitis.

We have evaluated the antibody and the effector T-cell responses to a single cerebrospinal fluid (CSF) infusion of myelin basic protein (MBP) in Lewis rats by measuring serum anti-MBP antibodies and clinical signs of experimental autoimmune encephalomyelitis (EAE), respectively. Some rats developed anti-MBP antibodies, but none manifested EAE in response to the primary infusion. Antibody responses to an EAE challenge 3 weeks after CSF infusion were normal, but clinical symptoms of EAE were markedly suppressed. Brain trauma at the time of MBP pretreatment enhanced this suppression. The CSF route of MBP administration is more effective in inducing suppression of EAE than peripheral routes.

Albumin outflow into deep cervical lymph from different regions of rabbit brain.

Dynamics and pathways of 125I-labeled albumin (RISA) outflow from brain to deep cervical lymph have been studied in anesthetized rabbits between 4 and 25 h after microinjection of 1 microliter RISA into the internal capsule or midbrain. Lymph from the jugular lymph trunks was collected for periods of 2-11 h. RISA was cleared from brain with half-times of disappearance from internal capsule and midbrain of 18.2 and 11.9 h, respectively. RISA was distributed in high concentration to subarachnoid arteries that supplied the tissue injection site; this was consistent with RISA drainage from brain via perivascular spaces. Outflow through lymph rose to a maximum value 15-20 h after tracer injection. Mean recovery of RISA from lymph over the 25-h collection period accounted for 22% of total loss from internal capsule and 18% from midbrain. This result compares with mean recoveries from caudate nucleus and cerebrospinal fluid of 47% and 30%, respectively [M.W.B. Bradbury, H.F. Cserr, and R.J. Westrop, Am. J. Physiol. 240 (Renal Fluid Electrolyte Physiol. 9): F329-F336, 1981]. These are minimal estimates of total outflow to lymph because of the 15- to 20-h delay in RISA passage from brain to lymph.

Extracellular volume decreases while cell volume is maintained by ion uptake in rat brain during acute hypernatremia.

1. Regulation of brain extracellular and intracellular water content, regarded as volume, and electrolytes in response to 90 min of hypernatremia has been studied in the cerebral cortex of rats under urethane anaesthetic. 2. Total tissue electrolytes and water were partitioned between extracellular and intracellular compartments based on measurements made in two series of experiments. In one, tissue samples were collected and analysed for total water, Na+, K+ and Cl-. In the other, tissue extracellular volume fraction, [Na+] and [K+] were measured in situ using ion-selective microelectrodes. 3. Osmotically induced water loss from cerebral cortex was less than that predicted for ideal osmotic behaviour, revealing a degree of volume regulation, and this regulation was associated with net tissue uptake of Na+, Cl- and K+. 4. Total water content was 3.77 g H2O (g dry weight)-1 in control cortex and this decreased by 7% after 30 min of hypernatremia and then remained relatively stable at this value. Control extracellular water content, based on an extracellular volume fraction of 0.18, was 0.88 g H2O (g dry weight)-1. Control intracellular water content, estimated as the difference between total and extracellular water contents, was 2.89 g H2O (g dry weight)-1. After 30 min of hypernatremia, extracellular water content decreased by an average of 27% but intracellular water did not change. This indicates selective regulation of cell volume. By 90 min the extracellular water content had decreased by 47% and the loss in extracellular water content appeared to be accompanied by a roughly equivalent increase in intracellular water content. The intracellular volume increase, however, was not statistically significant. The tortuosity of the extracellular space averaged 1.57 and increased to 1.65 during the hypernatremia. 5. Brain extracellular fluid and plasma [Na+] were roughly equal in control tissue. Both increased by 30 mu equiv (g H2O)-1 as a result of the hypernatremia, although extracellular [Na+] lagged behind the plasma value during much of the first 60 min of hypernatremia. Extracellular [K+] was homeostatically regulated at 3 mu equiv (g H2O)-1 independent of changes in plasma electrolytes. 6. Estimates of extracellular and intracellular ion content (mu equiv (g dry weight)-1) indicate that extracellular Na+, Cl- and K+ content decreased during hypernatremia, by 32, 21 and 42% respectively, whereas intracellular ion content increased by 100, 169 and 5% respectively. 7. It is concluded that during acute hypernatremia the extracellular space decreases in volume through the loss of water and electrolytes while the intracellular compartment maintains its water content and gains electrolytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Barrier membranes at the outer surface of the brain of an elasmobranch, Raja erinacea.

This report gives the results of the first electron-microscopic examination of the cell layers covering the outer brain surface and the inner surface of the cartilaginous skull in the skate, Raja erinacea. The perivascular glial blood-brain barrier--a characteristic of elasmobranchs--extends to the outer surface of the brain. This outer barrier layer is surrounded, in turn, by a subarachnoid compartment (depth: 30-40 microns), containing loose connective tissue and blood vessels; by an arachnoid-like epithelium (10-15 cell layers), impermeable to horseradish peroxidase; and, by perimeningeal fluid, a fluid with a slow turnover rate and a protein composition different from plasma. The inside of the skull, facing the perimeningeal fluid, is covered by a multilayered (10-15 layers) cuboidal epithelium, also impermeable to horseradish peroxidase. Closely apposed cells in the luminal layer of this epithelium have apical microvilli and numerous vesicular profiles, containing material of moderate electron density. These observations may explain, in terms of structure, the regulated protein content of perimeningeal fluid and the restricted exchange of solutes between brain and perimeningeal fluid in elasmobranchs.

Distribution of extracellular tracers in perivascular spaces of the rat brain.

Large molecular weight tracers (india ink or albumin labeled with colloidal gold, Evans blue or rhodamine) were micro-injected into the perivascular space of an artery or vein on the brain surface, or within the cerebral cortex or the subarachnoid space of anesthetized rats. The subsequent distribution was followed both under intravital microscopy, in order to outline the pathways and direction of tracer movement, and in histological section, in order to describe the pathways of flow at the light and electron microscopic level. The tracers remained largely in the perivascular spaces and in the interconnecting network of extracellular channels, including the subpial space and the core of subarachnoid trabeculae. Tracer also leaked across the pia into subarachnoid CSF. Bulk flow of fluid within the perivascular space, around both arteries and veins, was suggested from video-densitometric measurements of fluorescently labeled albumin. However, this flow was slow, and its direction varied in an unpredictable way. These results confirm that perivascular spaces may serve as channels for fluid exchange between brain and CSF, but do not support the idea that CSF circulates rapidly through brain tissue via perivascular spaces.

Role of cervical lymph nodes in the systemic humoral immune response to human serum albumin microinfused into rat cerebrospinal fluid.

The humoral immune response to human serum albumin (HSA) microinfused into cerebrospinal fluid (CSF) has been measured in serum, cervical lymph nodes, and spleen of Sprague-Dawley rats. Conditions were designed to promote normal brain barrier function. Serum titers of anti-HSA antibodies, primarily IgG, increased over 10 days and then persisted for at least 10 weeks. A significant role for cervical lymphatics in the systemic response to CSF-administered HSA is suggested, based on results showing that (1) cervical lymph obstruction reduces serum titers of anti-HSA antibodies, and (2) total antibody production by combined superficial and deep cervical nodes, sampled 14 days post-immunization, exceeds that by the spleen.

Brain ion and volume regulation during acute hypernatremia in Brattleboro rats.

Regulation of brain ions and volume in response to 30 min of hypernatremia has been studied in two strains of anesthetized rats, the vasopressin-deficient Brattleboro and its vasopressin-competent parent strain, the Long-Evans. Plasma [Na] was increased by intraperitoneal injection of hyperosmolal NaCl. Brain volume was regulated during hypernatremia associated with tissue uptake of Na and Cl in both strains, but osmotically stimulated uptake of Na was 61% less in the Brattleboro. Blood-to-brain transfer constants for 22Na, measured as a function of plasma osmolality, were similar in the two strains. In contrast, bulk flow of cerebrospinal fluid (CSF) into brain, induced by osmotic dehydration of brain, was 55% less in the Brattleboro. CSF secretion in unstressed animals was also reduced, by 34%, in the Brattleboro compared with the Long-Evans. Reduced Na uptake by the brain of the Brattleboro rat during hypernatremia can be explained on the basis of a three-compartment model of brain volume regulation. Results support a function for vasopressin in brain ion homeostasis.