Helen F. Cserr

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.

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 radiolabeled 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.

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.

Flow of cerebral interstitial fluid as indicated by the removal of extracellular markers from rat caudate nucleus.

Bulk flow of interstitial fluid (ISF) in brain was studied by following the intracerebral distribution of horseradish peroxidase (HRP) injected into the caudate nucleus of rats. Using this technique, channels of ISF flow are outlined as the extracellular pathways of protein distribution away from the injection site. Anionic isoenzymes of HRP were separated from Sigmas Type II HRP using ion-exchange chromatography. Anionic isoenzymes proved superior as markers of ISF flow, presumably because there is less cellular uptake and surface adsorption of anionic protein. Analysis of the distribution of protein 4–8 hr after injection suggests that ISF flows from narrow intercellular clefts of the neuropil along a system of extracellular pathways including perivascular and periventricular areas and between fiber tracts. From this system ISF appears to drain into cerebrospinal fluid (CSF) and possibly also into fenestrated vessels within the brain. Quantitative aspects of fluid drainage from brain were investigated by measuring the rate and route of efllux from brain of radiolabelled extracellular markers: [ 3 H]polyethylene glycol (PEG: 4000 daltons) and [ 14 C]dextran (70 000 daltons). In nine control rats, the amount of isotope remaining in brain 4 hr after injection into the caudate nucleus, as percent of the injected dose (mean± S.E.M. ), was 55·8±6·3 for PEG and 63·1±6·6 for dextran. Similarity in the rates of dextran and PEG removal from brain, despite large differences in molecular weight, is consistent with removal from brain by bulk flow of ISF. A maximum of 20% of the isotope cleared from brain could be recovered from CSF, in agreement with anatomical evidence that CSF may not be the sole route of ISF removal from brain.

Bulk flow of interstitial fluid after intracranial injection of Blue Dextran 2000

Abstract The hypothesis that there is bulk flow of cerebral interstitial fluid (ISF) has been examined in rats by following the intracerebral distribution of Blue Dextran 2000. Guide tubes were implanted into the caudate nucleus using standard stereotaxic technique. One day after implantation, 0.5 μl of saline containing Blue Dextran was injected into brain. The extent and pattern of dye distribution were determined 15 min and 24 hr after injection by microscopic examination of brains sectioned using a freezing microtome. Blue Dextran was initially confined to the base of the injector cannula, but spread extensively over 24 hr. Characteristics of dye distribution indicate that Blue Dextran is transported away from the injection site by bulk flow of cerebral ISF, possibly along the course of cerebral blood vessels. There was no evidence of bulk flow in rats pretreated with dexamethasone or with an injection-implantation interval of 7 days, suggesting that edema fluid contributes to the observed flow of ISF. Results indicate the need to reevaluate the role of perivascular spaces in net fluid exchange between brain and cerebrospinal fluid.

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.

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)

Physiology and immunology of lymphatic drainage of interstitial and cerebrospinal fluid from the brain

Cerebrospinal fluid (CSF) and brain interstitial fluid, extracellular fluids produced in the central nervous system (CNS). efflux by several routes (Figure 1) [reviewed in 4, 51. A major outflow pathway for extracellular fluid is passage through arachnoid villi into blood of the dural sinus. Another efflux pathway is drainage into lymph along routes adjacent to cranial and spinal nerves and leads to regional lymph nodes [15]. Following injection of radiolabelled protein into the CNS, a significant fraction (14-47%) of this tracer leaving the CNS appears in cervical lymph (Table 1). Furthermore, protein injected into the CNS reaches the cervical lymph in a relatively higher concentration compared with protein injected into the blood. Drainage to cervical lymph is dependent upon several factors, including site of injection into brain, animal species, and assay time period (Table 1). Drainage along the olfactory route, through the cribriform plate, and into cervical lymph is a significant pathway in animals [l]. Both blood and lymph efflux pathways are accessible to cells as well as to macromolecules. Thus, passage of these constituents to the spleen and draining lymph nodes raises interesting issues concerning their immunogenicity. Classical observations that allografts survive for more extensive time periods when implanted into brain (compared to peripheral sites) have led to the concept of immune privilege of the brain [2]. In our laboratory, we have developed a model in rats to analyse the immunological significance of antigen outflow from the CNS [8]. An important feature of this model is that a

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.

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.