CNS changes in hyperbilirubinemia. Functional implications.

Hyperbilirubinemia is a recognized etiologic factor in motor and hearing disorders associated with cerebral palsy. Its role in more subtle forms of neurological impairment is more controversial. Using a mutant animal model, which develops symptoms and signs closely resembling the human kernicterus syndrome, neurons of hippocampus, cerebral cortex, cochlear nuclei, losuc ceruleus, and olfactory bulb were examined by electron microscopy. Pathological changes, observed in all areas studied, consisted of mitochondrial and endoplasmic enlargement and vacuolation, with glycogen deposition; increased extracellular space; myelin figures; and degenerating changes in nerve terminals. If we make the assumption that pathologic changes in the human infant with neonatal jaundice are similar to changes in the animal model, then the widespread involvement of CNS neurons in all cortical areas examined may well help to explain the syndromes of minimal cerebral dysfunction reported in clinical studies.

The terminal innervation patterns in young and old guinea pig heart valves: a quantitative analysis using acetylcholinesterase staining.

The objective of this study was to determine whether and to what extent age-related changes occur in atrioventricular (AV) heart valve innervation. The AV valves from three young adult (3 months) and three older (> 24 months) female guinea pigs were studied. An acetylcholinesterase (AChE) localization method was used to prepare valve whole mounts for analysis. Two methods were used to assess nerve fiber density. Segments of the valves were drawn using a camera lucida/Nikon optiophot system. The density of nerve fibers was calculated from digitized images. The density of nerve fibers was also calculated by counting the points at which the nerve plexus intersected with the grid lines of an ocular graticule. In the bicuspid and tricuspid valves of the older guinea pigs, we observed a marked diminution in the densities of the nerve plexus, particularly in the basal zone, towards the free edges of the valve cusps, and in the chordae tendineae. Whole mount preparations such as those used in our morphological studies of the AV innervation may assist in elucidating the changes in other autonomic nerve plexuses with aging. Further work is required to establish whether and to what extent the loss of valve innervation influences the effectiveness of closure of the valves.

Neurotensin immunoreactivity in the central nucleus of the rat amygdala: an ultrastructural approach.

Neurotensin (NT) was demonstrated in the central nucleus of the rat amygdala (CNA) using a modification of the avidin-biotin complex immunohistochemical technique. Electron-dense reaction product (particles were 15-25 nm in diameter) was localized in perikarya, dendrites, axons, and axon terminals. It was found also associated with profiles of rough endoplasmic reticulum, mitochondria, microtubules, and small agranular as well as large granular vesicles. In distal dendrites, the reaction product was associated with microtubules, vesicles, and postsynaptic densities. Axon terminals of three types formed synaptic contracts with NT-immunoreactive neurons in the CNA: one was characterized by numerous round or oval agranular vesicles, the second by numerous pleomorphic vesicles, and the third by agranular vesicles that were loosely distributed and pleomorphic. All three types formed symmetric axosomatic and asymmetric axodendritic contacts. NT-immunoreactive axon terminals containing small round agranular vesicles stood out clearly from the intermingling profiles of immunonegative structures. We found numerous glomeruli, each consisting of a central NT-immunoreactive dendrite surrounded by all three types of axon terminals. We observed that some NT-immunoreactive terminals formed symmetric axoaxonal contacts with each other, providing evidence for the presence of local NT-to-NT circuits, whereas many others synapsed with axon terminals devoid of NT immunoreactivity.

The role of substance P-containing fibers in sympathetic ganglia: effect of capsaicin.

Capsaicin was given subcutaneously to guinea pigs and the effect on substance P-immunoreactive (SP-I) fibers in the celiac/superior mesenteric and inferior mesenteric ganglia was observed at 2 day and 8-10 day intervals. Capsaicin (125 mg) treatment led to almost total disappearance of SP-I fibers from all areas examined in both short- and long-term animals. This effect applied equally to the dense network of varicose SP-I fibers and to basket-like SP-I contacts with principal ganglionic neurons. The effect of capsaicin on SP-I fibers in the mesenteric ganglia provides a strong indication that these fibers represent a homogeneous population of visceral sensory afferents. This is supported by other lines of anatomical evidence in the literature. Taken together with studies that have shown axodendritic contact of SP-I terminals on principal ganglionic neurons and neuro-modulatory effects of SP on these neurons, it may be hypothesized that SP-I fibers in the mesenteric ganglia represent collaterals of visceral sensory afferents forming a subspinal feedback arc.

Ultrastructural evidence for remodelling in a central noradrenergic pathway following electrolytic lesioning.

Electrolytic lesions were carried out in the medial hypothalamus of adult rats to study remodelling responses in a central noradrenergic pathway, the medial forebrain bundle. Four days, two weeks, 4 weeks and 8 weeks post-lesion, the animals were perfused and processed for correlated fluorescence microscopic (FM) and electron microscopic (EM) study. FM evaluation 4 days post-lesion showed that, compared with control preparations, catecholaminergic fibers became thick, distorted and intensely fluorescent. With increasing survival times the caliber of these fibers became finer and fluorescence intensity was gradually diminished. Some of the small blood vessels in the vicinity of the lesion acquired an intense perivascular fluorescence. Electron microscopic examination of the lesion site 4 days post-lesion disclosed many degenerating axons and increased extracellular space. No increased extracellular space was discerned by 8 weeks post-lesion. After all survival periods greatly enlarged axonal profiles were seen, and these resembled 'growth cones' described in earlier tissue culture, developmental and peripheral nervous system studies.

Plasticity of catecholaminergic terminals in rat paraventricular hypothalamic nucleus after 6-hydroxydopamine lesion: an emphasis on bouton sizes and synaptic frequency.

Catecholaminergic (CA) nerve terminals in the paraventricular hypothalamic nucleus (PVN) of adult rats were studied at 4, 21, 56 and 180 days after a single injection of 6-hydroxydopamine (6-OHDA) neurotoxin into the right lateral ventricle of the brain. We previously described and quantified the extent of CA terminal sprouting in the PVN after 6-OHDA lesions. For this communication we studied parameters, specifically the bouton sizes and the synaptic frequencies of CA terminals during the renewal process, and evaluated how changes of these parameters are related to axonal sprouting. The CA boutons were identifiable in the electron microscope by exhibiting small granular vesicles (SGVs) after central administration of 5-hydroxydopamine (5-OHDA) marker. The marked CA boutons were measured and further categorized according to whether or not they were associated with distinct synaptic specializations at various post-lesion stages. The average sizes of CA boutons were strikingly similar in their diameters (1.0 micron) for both control and experimental tissues. However, CA boutons larger than 2.1 micron were rare and seen more often in the experimental tissues with 6-OHDA lesion and were sustained up to 180 days after lesions. Catecholaminergic profiles with ultrastructural features of growth cones were also seen in the PVN following the 6-OHDA lesions, indicating that there is growth activity in the PVN after 6-OHDA lesion. There were 33% of CA boutons in the PVN from the control tissues that appeared to have synaptic contacts.(ABSTRACT TRUNCATED AT 250 WORDS)

Collateral sprouting of somatostatin-immunoreactive axons after partial deafferentation of the central nucleus of the rat amygdala.

These experiments utilize a paradigm developed to study plastic responses of peptidergic neurons in a discrete brain area following deafferentation. The central nucleus of the amygdala (CNA) is richly innervated by somatostatin-immunoreactive (SS-I) terminal axons. In the course of preliminary light microscopic (LM) investigations by this laboratory, changes were observed in the density of presumed SS-I terminals in the rat CNA after lesioning the medial input. The LM finding of increased density of presumed SS-I terminals in the CNA at the 10-day post-lesion stage underscored the need for a quantitative electron microscopic (EM) study of the SS-I components, including an evaluation of synaptic events at different survival periods. At the 3-day post-lesion stage, EM examination showed degenerating axons in the lesioned CNA, many already engulfed by astrocytes. None of the degenerating profiles were SS-I, supporting the view that the lesion did not interrupt, to any significant extent, SS-I axons entering the nucleus. EM surveys of the 10-day post-lesion material demonstrated that degenerated profiles had almost completely disappeared. Numbers of SS-I axon terminals, particularly of smaller-sized profiles, were increased by 22% over control value. Synaptic frequency was decreased by 16% below control value. Numbers of SS-I terminals making synapses were increased 3.4% above control value. At the 30-day post-lesion stage, the total number of SS-I terminal axons had increased 86% over controls, whereas the synaptic frequency had decreased by about a third below controls. The absolute number of SS-I terminals engaging in synapses had increased by 24% over controls. The 90-day post-lesion CNA showed a further increase in the number of SS-I axon profiles: 136% over control value. The synapse-to-axon ratio (synaptic frequency) of 27% was similar to that observed for the CNA from the unlesioned side or from unoperated animals. At this stage the number of SS-I synapses had increased by 135% over controls. This model presents many possibilities for studying neuroplasticity, particularly involving peptidergic neurons of the central autonomic nervous system.

Bradycardia following injection of 6-hydroxydopamine into the intermediate portion of nucleus tractus solitarius medialis.

Studies were carried out to determine the effects of selective destruction of the catecholamine innervation of the intermediate portion of the nucleus tractus solitarius medialis (NTSm) on mean arterial pressure and heart rate in conscious rats. Following bilateral injections of 6-hydroxydopamine into the intermediate portion of NTSm, animals demonstrated a prolonged bradycardia, but no changes in mean arterial pressure or mean arterial pressure lability, when compared with controls. Results indicate that the baroreceptor reflex loop which mediates bradycardia is sensitive to impulses from catecholamine axons entering the intermediate NTSm.

Catecholamine inputs to the nucleus tractus solitarius.

The projections of brainstem catecholaminergic (CA) cell groups to the rat nucleus tractus solitarius (NTS) were examined using 6-hydroxydopamine (6-OHDA) injections and glass microknife cuts. 6-OHDA (4 micrograms) was injected into the intermediate NTS, and this resulted in depletion of CA fluorescent varicosities from the NTS at this rostrocaudal level, except for varicosities along the periventricular edge of the NTS. In addition, a band of swollen fluorescent axons extended between the CA A1 cell group of the ventrolateral medulla and the lateral NTS. Microknife cuts were used to interrupt the projections of the CA A1 and A2 cell groups (located in the caudal NTS) and tissues were examined for changes in CA varicosity density within the intermediate NTS. Following transverse knife cuts of the intermediate NTS, rostral to the A2 cell group, fluorescent varicosities rostral to the cut virtually disappeared, and the fluorescence intensity of the ipsilateral A2 neurons caudal to the cut was increased. These cuts also eliminated the 6-OHDA-resistant varicosities along the periventricular NTS. After microknife cuts lateral to the intermediate NTS, the fluorescent varicosity density in the NTS was unchanged. These results indicate that the major CA projection to the NTS arises from the ipsilateral A2 cell group. The 6-OHDA-resistant varicosities arising from neurons caudal to the knife cut probably arise from the adrenergic C2 cell group.

Tachykinins in the canine gastroesophageal junction.

Lower esophageal sphincter (LES) effects produced by the mammalian tachykinins were evaluated in anesthetized dogs. The distribution and content of substance P (SP) and neurokinin A (NKA) in the region of the canine gastroesophageal junction was also studied. SP and NKA stimulated a linear dose-dependent contraction of the LES after intra-arterial administration. Neurokinin B (NKB) failed to stimulate an increase in LES pressure (LESP). SP was characterized by an immediate but short-lived contraction followed by a period of relaxation. NKA stimulated a potent LES contraction that was slow in onset but long-lasting. On an equimolar basis, both SP and NKA were approximately 100 times more potent LES stimulants than bethanechol or phenylephrine. Pretreatment with atropine (muscarinic blockade) or tetrodotoxin (neural blockade) inhibited the effect produced by SP. NKA appeared to stimulate LES contraction independent of neural or cholinergic mechanisms. Radioimmunoassay revealed a regional variation in tachykinin content in the gastroesophageal junction. Ganglia, cell bodies, nerve fascicles, and neurites stained specifically for both SP and NKA. The variable effects, potencies, and mechanisms of action observed in this study suggest the presence of specific tachykinin receptor subtypes in the gastroesophageal junction. Both SP and NKA were found to have a broad neural distribution in this region. These findings suggest that the tachykinins may play an important role in neuroregulation of LES smooth muscle.

An improved method for perfusion fixation of neural tissues for electron microscopy.

A method employing vascular perfusion for improved preservation of biological ultrastructure is described, and its effectiveness demonstrated for mammalian nervous tissues. Following a physiological saline flush into the aorta, hydrogen peroxide and glutaraldehyde in phosphate buffer are perfused. After buffer rinses, tissue blocks are postfixed in osmic acid and potassium ferrocyanide. The success rate is enhanced greatly by close attention to details of perfusion technique. Advantages of the method include more uniform and complete preservation. In particular, superior images of membranous elements, glycogen granules and basal laminar material are achieved. Adjustments in osmolality may render the procedure suitable for nonmammalian forms and other tissues.

Is the mitral valve passive flap theory overstated? An active valve is hypothesized.

The concept that the mitral valve of the heart is a passive flap that opens and closes like a barn door has been emphasized for decades by medical and biology professors to their students. But experimental findings, which are outlined in this report, support the theory of an active valve. We hypothesize that the two leaflets of the mitral valve are actively contractile; that physical forces generated in the valve itself may stabilize and add precision to the sum of forces that regulate valve movement. This precision could be of critical significance both in the moments preceding, and during, valve opening and closing. Evidence supporting our active valve hypothesis includes the profuse innervation of motor and sensory nerves that are present in the mitral valves of all animals studied. In addition, multiple contractile cell types have been found in the mitral valve, including cardiac muscle cells, smooth muscle cells, and cardiac valvular interstitial cells. In vitro work in our laboratories using the rat mitral valve shows that not only are the valves capable of contraction and relaxation, but that the contractions and relaxations are nerve-mediated. We theorize that the rich innervation and contractile cells in the mitral valve work together to modulate fine-tuning of valve movements and tone, thereby ensuring the integrity of the valve seal. Other investigators have reported that the mitral valve demonstrates contractile activity and that denervation localized to the mitral valve affects valve competence. The evidence for an active mitral valve presented by these and other experimental studies warrant a reexamination of the validity of the passive valve concept. An accurate and full understanding of the precise movements of the valve leaflets and the mechanisms that regulate these movements is likely to provide the information needed to understand and develop treatments for many different cardiac valve problems, including mitral valve diseases such as prolapse and myxomatous degeneration. In view of the available experimental evidence, the concept that the mitral valve functions only as a passive structure is challenged by numerous anomalies. A reinterpretation of the concept of valve function that incorporates active as well as passive roles for the valve leaflets and other components of the valve apparatus would have significant implications both for the directions taken in research involving the cardiac valves and for the approaches to treatment.

Alterations of the intestinal innervation in mice infected with Schistosoma mansoni.

Schistosomiasis mansoni is a parasitic disease in which granulomas form around schistosome eggs in the liver and intestines. The purpose of this study was to determine the alterations in the intrinsic innervation of the distal ileum and proximal colon resulting from schistosomiasis. Using murine schistosomiasis mansoni, we examined light microscopic preparations stained with osmium-zinc iodide or the dihydronicotinamide adenine dinucleotide: nitro BT oxidoreductase (NADH) method. We also examined specific populations of peptidergic nerves (vasoactive intestinal polypeptide and substance P) using an avidin-biotin complex (ABC) immunohistochemical technique. We found that granulomas focally destroyed the enteric nerves. Occasionally nerves were found within granulomas, particularly at the periphery of the lesions. Nerve cell bodies close to granulomas had altered staining, which included increased staining for vasoactive intestinal polypeptide. The distribution of nerve injury varied between the 2 enteric segments studied. In the distal ileum, the principal injury was to the myenteric plexus; whereas, the submucous and mucosal plexuses were predominantly damaged in the proximal colon. The physiologic significance of this injury to the enteric nerves requires elucidation.

Histofluorescence and ultrastructural observations of small intensely fluorescent (SIF) cells in the superior sympathetic ganglion of the guinea pig.

Amine-containing small intensely fluorescent (SIF) cells are ubiquitous in vertebrate sympathetic ganglia and, in some species, SIF cells have been identified as interneurons. The hypothesis proposed in this study is that SIF cells in superior sympathetic ganglia of the guinea pig function as interneurons, with efferent connections characteristic for the species. Fluorescence (catecholamine) microscopy and 5-hydroxydopamine marker for electron microscopy were used to study SIF cells, their processes and connections in this ganglion. Brightly fluorescent fibers were seen attached to virtually all SIF cells, and were of two types. The first type, single or arranged in cords, interconnected elements of the SIF-cell system; these apparent linkages joined individual SIF cells as well as adjacent clusters. The electron-microscopic evidence for synaptic contacts between SIF cells warrants the claim that integrated action is a presumed function of these elements. The second type of SIF-cell process was generally of greater length. These individual, branching fibers made presumed connections with dendrites of most principal ganglionic neurons. This arrangement suggested by histofluorescence preparations was confirmed by electron microscopy to involve synaptic connections, and the postsynaptic element was shown to be continuous with the perikaryon of the principal ganglionic neuron. Ultrastructural evidence that collections of dense-cored vesicles occur within processes of both principal ganglionic neurons and SIF cells, in proximity to unsheathed portions of plasma membrane, leads to the conclusion that interstitial diffusion of catecholamine from both may occur; the finding of SIF cell processes adjacent to fenestrated blood vessels suggests that catecholamine may also be transported through capillaries.

Ultrastructural aspects of bilirubin encephalopathy in cochlear nuclei of the Gunn rat.

The cochlear nuclei of homozygous Gunn rats aged 2 days to 7 months were examined. Ultrastructural abnormalities were observed in all age groups studied, including 2 and 4 days old animals. Mitochondrial alterations are amont the earliest manifestations of bilirubin encephalopathy (2 days). In mitochondria of large neurons, vacuoles were found which contained increasing (with age) collections of alpha and beta glycogen particles. Some of the larger 'ex-mitochondrial sacs' appear to have been caught at the point of disruption, with glycogen-filled vacuoles in close proximity. Dilated profiles of rough ER also contained glycogen particles. In the cytoplasm of the same large neurons, elaborate myelin figures surrounded tongues of cytoplasm, vacuoles and degenerative elements. Reconsideration of previous morphological and biochemical observations in the light of the present findings makes it appear very likely that bilirubin primarily affects membrane function, especially in mitochondria.

A quantitative histological study of changes in neurons and glia in the Gunn rat brain.

The rostral part of the anterior limb of the anterior commissure (RAL) and the indusium griseum of the brains of Gunn rats and Wistar rats were examined quantitatively to assess the effects of hyperbilirubinaemia on the developing nervous system. The number of glia in the RAL was significantly less in Gunn rats than in the Wistar rats. The number of glia in the indusium griseum was similar in both groups but the Gunn rats showed a decrease of about 20% in the number of neutrons in this region. Myelination appeared to be unaffected and apart from an apparent increase in the number of dense bodies in astrocytes and oligodendrocytes there were no obvious morphological changes in either glia or neurons.

Innervation of the mitral valve is strikingly depleted with age.

Previous reports demonstrated that mammalian atrioventricular (AV) valves possess a dense nerve plexus, consisting of nerve subpopulations which differ from each other in densities and patterns of distribution in the valves, and which may have sensory or motor roles in valve function. Although there is extensive evidence that age-related changes occur in autonomic nerves of animals and humans (Daly et al. J. Pharm. Exp. Ther., 1988;245(3):798-803; Ingall et al. Aust. NZ J. Med., 1990;20:570-577; Tumer et al. Exp. Gerontol., 1992;27:301-307), and that these changes contribute to changes in cardiac function (Klausner and Schwartz Clin. Geriat. Med., 1985;1(1):119-114), there is little information about age-related changes in heart valve innervation. In this study, we used acetylcholinesterase (AChE) histochemistry to localize and compare qualitative and quantitative changes in the innervation of the mitral valves in young adult and aged animals of three species. Young adult and aged guinea pigs, mice, and Wistar and Fischer 344 rats were anesthetized with Nembutal, the hearts removed, and the mitral valves dissected out and processed for AChE localization. Camera lucida drawings of the AChE-positive nerves in representative segments of valve cusps were made directly from slides; these drawings were digitized and subjected to computer-assisted image analysis to obtain quantitative information about nerve plexus density in the valves. All three animal species showed profuse AChE-positive innervation in the mitral valves of young adult animals, and decreases in the density of this innervation in aged animals. The most striking loss of innervation, compared to the young adult, occurred in the mitral valves of aged Fischer 344 rats, in which large regions of the valves appeared virtually devoid of nerves. Further studies are needed to investigate whether and to what extent age-related losses in heart valve innervation affect valvular structure and function.

Hypertrophy of rat sensory ganglion neurons following intestinal obstruction.

BACKGROUND: Neuroplastic changes following ileum hypertrophy have been reported in intrinsic enteric neurons. The hypothesis in the present study was that intestinal hypertrophy induces neuronal changes in dorsal root ganglia (DRG). METHODS: Under sodium pentobarbital anesthesia, partial obstruction was produced in the rat by tying a plastic ring around the terminal loop of ileum. Fast Blue (FB) (Sigma, St. Louis, MO) was injected into the obstructed ileum wall, and the rat was perfused after 8 days. DRG were immunostained and examined to identify and measure sizes of perikarya containing FB and/or calcitonin gene-related peptide (CGRP) or FB and/or substance P (SP). RESULTS: Of the DRG neurons that projected to the ileum in control or obstructed animals, approximately 50% were CGRP-immunoreactive (IR) and 30% were SP-IR (colchicine pretreatment was not used). Neurons that projected to the obstructed ileum were increased in size compared with neurons in nonobstructed controls. Some of these neurons were CGRP-IR or SP-IR; some were large FB-labeled neurons that were not SP-IR or CGRP-IR. CONCLUSIONS: The morphology of sensory autonomic neurons in adult animals is influenced by dynamic interactions with the targets they innervate, whether directly or transneuronally.

Tyrosine hydroxylase- and nitric oxide synthase-immunoreactive nerve fibers in mitral valve of young adult and aged Fischer 344 rats.

Using confocal fluorescence microscopy we studied, in whole mounts of heart mitral valves of young adult and aged Fischer 344 rats, the distribution of nerves containing the catecholamine marker tyrosine hydroxylase (TH) or the synthetic enzyme marker for nitric oxide, nitric oxide synthase (NOS). TH-IR was localized in two separate nerve plexuses which do not intermingle. The 'major' plexus arose from the annulus region, traversed the basal zone of the valve, and ramified in the intermediate zone to form a dense network of fine fibers. The 'minor' plexus was restricted to the distal zone and originated from bundles that ascended the chordae tendineae to enter the valve cusp. A concentric zone located between the major and minor plexuses was devoid of TH-IR nerve fibers. Both plexuses demonstrated (i) nerves that contained numerous varicosities along the length of each fiber, (ii) many terminal axons and (iii) different shaped terminal axon endings. With age, the density of TH-IR innervation in the mitral valve was markedly reduced; and nerve fibers of the minor plexus were limited to the chordae tendinae, without extending into the valve cusp itself. NOS-IR fibers in the mitral valve formed a loose network that extended from the annulus to more than halfway down the cusp. The varicose beads of the terminal NOS-IR axons appeared to become progressively smaller and less intensely fluorescent until they disappeared at the terminal endings, which showed no specializations. No NOS-IR fibers were observed in the distal zone of the valve leaflet or in the chordae. In the aged mitral valve, the density of NOS-IR nerves was decreased, as compared with NOS-IR innervation in the young adult valve. The existence of TH and NOS as well as other signal molecule markers in heart valve nerves and the disparate patterns of their distribution and localization provide evidence supporting the theory that heart valve nerves form a complex reflexogenic control system in the mitral heart valve. In summary, two distinct neural architectures are described for TH-IR and NOS-IR valve nerves, respectively. The former are believed to be axons dedicated to sympathetic motor functions. The NOS-IR valve nerves may have sensory and/or postganglionic parasympathetic motor functions. An implication of these findings is that different, but perhaps related, valve functions may be mediated by separate, dedicated circuits.

Effect of surgical trauma on the endogeneous heme iron in the brain.

Paucity of electron-microscopic studies on the fate of endogenous heme iron, released as a result of planned surgery, in the central nervous system warranted the present work. During investigations of neuronal plasticity in the cat, an experimental model, in which the proximal cut end of the vagosympathetic trunk was rerouted into the diencephalon, presented an ideal substrate for studying this problem. At 3 months, the brain tissue containing the implant was processed for light and electron microscopy and histochemistry. Under light microscopy, Prussian-blue-positive, dense cytoplasmic aggregates were visible within the macrophages. The cells were mostly clustered around the junctional area between the central and peripheral nerve elements. Electorn-microscopic findings were highly characteristic and distinguished the macrophages from the surrounding neural elements by their rich content of electron-dense granules. While the latter were mostly scattered as free cytoplasmic particles, large aggregates of the same were also observed inside the lysosomes. Besides, a few inclusions were seen within astrocytic processes, close to the plasma membranes. At higher magnifications, the particles revealed the characteristic internal subunit structure of ferritin micelles. The uptake of hemoglobin and its subsequent conversion of ferritin within the macrophages and astrocytic processes are discussed at subcellular level in the light of other available studies.