INHAND: Non-proliferative and proliferative lesions of fish – Chapter 8. Fish nervous system

Introduction

The vertebrate central nervous system (CNS) consists of brain and spinal cord. It is an evolutionarily conserved system, so that embryogenesis leads to partitioning of all vertebrate brains into forebrain (prosencephalon), midbrain (mesencephalon) and hindbrain (rhombencephalon). Forebrain consists of telencephalon and diencephalon. Hindbrain consists of the metencephalon and myelencephalon, with the transition to the spinal cord. In higher vertebrates, like humans, non-human primates and rodents, the telencephalon expands substantially and envelops both the diencephalon and mesencephalon and becomes the cerebral cortex or cerebrum with complex histological features¹.

Although basic regions of the teleost brain are similar to mammalian brains, there are significant differences in forms and complexity. The adult brain of fish, reptiles, amphibians and birds exhibits a simplified architecture with three major subdivisions of the brain (fore-, mid- and hindbrain), which remain situated along the anterior-posterior axis of the vertebra. Among fishes, even though they share basic characteristics of the central and peripheral nervous systems, there are notable variations in anatomical and histological features that are unique and different when compared to higher mammals as well as between different species. For example, white matter and grey matter cannot be easily differentiated in some species. There are also differences in development of the fish brain when compared with the mammalian. The teleost telencephalon develops by eversion while the mammalian brain develops by evagination². Because fish like zebrafish are used extensively as models for human development and diseases, subtle differences in the development and histology of specific fish species are important for these sophisticated models and their appropriate application to humans in health and disease.

In addition, fish white matter matter is relatively less dense compared with white matter in higher vertebrates. In fish and lower vertebrates, the telencephalon is a considerably smaller structure situated anterior to the mesencephalon and from which the more prominent olfactory bulb projects.

In this section on the nervous system, introductory material includes a review of comparative interspecies anatomy differences, followed by tabulated microscopic findings in a standardized format.

Morphology

Neurons and neuroglia are the two primary classes of functional cells within nervous system. The nervous system is classically divided into central nervous system (CNS) and peripheral nervous system (PNS)^{3, 4}. Generally the PNS of fish is similar to mammals, whereas there are far more structural differences between the CNS of fish and mammals. The CNS architecture is classically divided into telencephalon, diencephalon, mesencephalon, rhombencephelon (metencephalon, myelencephalon) and medulla spinalis. The most rostral part of the brain is the paired olfactory bulb. The telencephalon is responsible for olfaction and for aspects of memory, reproductive behavior, feeding behavior, and color vision⁵.

The diencephalon has five major subdivisions including epithalamus, dorsal thalamus, ventral thalamus, posterior tuberculum, and hypothalamus. The mesencephalon includes optic tectum (dorsally) and torus semicircularis (ventrally).

Rhombencepelon (hindbrain) is divided into rostral metencephalon and a caudal myelencephalon. This includes cerebellum and medulla oblongata. The cerebellum, or metencephalon, is associated with reception and coordination of proprioceptive and balance stimuli.

There are notable species differences in the fine details of the structure and function of the blood brain barrier between different fish species, but basic anatomic structures and functions among teleosts tend to be consistent. The teleost meninges (endomeninx) comprises outer, intermediate and inner layers attached to the brain; this may be considered similar to the leptomeninges in mammals^{3, 6}. The cells of the intermediate endomeningeal layer are morphologically similar to specialized endothelial cells in that they contain well developed junctional complexes that constitute a barrier to meningeal diffusion in addition to their role in active transcellular transport. Ectomeninx is an extracellular matrix tissue that contain collagen, collagen and mucin admixed with blood vessels⁶.

Despite the considerable neuroanatomical differences between the mammalian and teleost CNS, mounting evidence shows key homologous functions in several zebrafish CNS areas compared with similar structures in mammals⁷. For example, lateral pallium of telencephalic area of zebrafish is responsible for memory processes, whereas the habenula is associated with fear responses, similar to the hippocampus and the amygdala in mammals, respectively. Additional information can be found in Hugh Ferguson’s Systemic Pathology of Fish, 2^nd edition and other publications (see recommended reading in introduction, doi: 10.1293/tox.2025-0191).

Physiology

The diagnostic nomenclature of the nervous system is based on the cell type that is damaged, and on the specific cellular components that are altered in those locations. Neurons are the chief functional elements of the CNS and of the ganglia in the PNS. A typical neuron has a cell body (perikaryon), multiple dendrites (that receive incoming signals), a single axon (that carries outgoing signals), and presynaptic terminals. Neurons may be large like the motor neuron or Purkinje cells, or small like cerebellar granule cells. Glial cells consist of astrocytes, oligodendrocytes (CNS) and Schwann cells (PNS) which constitute macroglia. Microglia are the resident macrophage cells of the CNS, which are derived from mesodermal precursors of the hematopoietic system. They seed the CNS prior to formation of the definitive hematopoietic tissues and participate in immune surveillance and phagocytosis within the CNS^{8, 9}.

Generally, fish rapidly respond to injury of neuronal cells with astrocytes and microglia^{8, 10}. Their rapid phagocytic activity and secretion of growth-promoting factors contribute to a tissue environment that promotes robust regeneration by optic axons^11,12,13. This concept of the greater ability of CNS tissue to regenerate in fish compared to mammals is very important as is the related observation, that, because there are high numbers of stem cells in the brains of adult fish, tumors observed in the adult piscine brain tend to resemble those of pediatric humans rather than adult humans^{14, 15}. In particular, a number of studies in the C. auratus CNS demonstrated a large microglial response to Wallerian degeneration after optic nerve section, supporting that the majority of reactive cells in the visual path after axotomy are local microglia^{12, 16, 17}. Due to presence of numerous stem cells, teleost fish exhibit an enormous potential to produce new neurons in the adult central nervous system and to replace damaged neurons by newly generated ones¹⁸.

Necropsy, Fixation and Trimming

Following routine sacrifice, it is recommended that dissection of the brain and/or spinal cord not be attempted immediately. Instead, the dorsal calvarium is removed, and the brain is preserved and fixed in situ for 24–48 hours prior to excision. Special care must still be taken to avoid damaging the delicate fish brain tissue while separating from the skull. As an alternative to brain or spinal cord excision, CNS tissues may be examined in situ in small or juvenile fish that are preserved and sectioned as whole body or head specimens, in which case it is still prudent to excise the dorsal calvarium to promote fixative penetration. Although it is possible to use 10% neutral buffered formalin (NBF) or alcohol as initial fixatives, superior results with fewer artifacts are often achieved by initial fixation in Davidson’s, Dietrich’s, or Bouin’s solutions for 24–48 hours, followed by a thorough rinse with seventy percent ethanol, after which the tissues may be maintained in NBF prior to processing. It is additionally possible to perfusion fix the brains of even small species such as zebrafish, although this approach is used primarily for specialized research projects rather than routine toxicology studies. For this procedure, fish are deeply anesthetized with methane sulfonate salt, after which they receive cold phosphate buffered saline (PBS) solution followed by cold Alcohol Formalin Acetic Acid (AFA) fixative (90 ml: 80% ethanol, 5 ml: 37% formalin, 5 ml: glacial acetic acid) by transcardiac perfusion. The perfused fish are then euthanized by decapitation, and the skull is opened dorsally to expose the brain. Excised brains or whole fish may be embedded for sectioning in the sagittal, transverse, or frontal planes, depending on the primary anatomic region(s) of interest. If the anatomy of the particular fish species is unfamiliar, it may also be advisable to section the brain in the same orientation as can be found in species-specific reference materials.

Brain, Spinal Cord and Nerves – Congenital Lesions

Congenital lesions of the nervous system are not typically observed in toxicological studies that involve fish. However, they may be observed in specific instances, such as when used as a model to study early development using large-scale mutagenic screens, systems toxicology screenings, developmental neurotoxicity testing or as a model to study human congenital disorders that result in various CNS anomalies^19,20,21.

Table 1 shows nomenclature, diagnostic criteria, and differential diagnosis of brain, spinal cord and nerves.

Table 1. Nomenclature, diagnostic criteria and differential diagnosis brain, spinal cord and cranial nerves.

Brain, spinal cord and nerves			Common	Uncommon	Not observed but potentially relevant	Not Applicable
Congenital/Developmental
	Cyst, squamous				X
	*Ectopic Tissue			X
	Hamartoma, lipomatous				X
	Heterotopia, neuron				X
Non-proliferative
	Neuron-cell body
		*Cellularity decreased, neuron		X
		Chromatolysis		X
		*Fungus		X
		*Necrosis, neuron		X
		*Neuronophagia		X
		Parasite		X
		*Vacuolation, neuron		X
	Neuron- axon
		*Atrophy, axon		X
		*Degeneration, axon		X
		Dystrophy, axon		X
	Glia – cell body
		Type II astrocytes			X
		Astrocyte swelling/vacuolation			X
		Astrocytosis		X
		*Gliosis		X
		*Microgliosis		X
		*Satellitosis		X
	Glia - myelin
		Demyelination		X
		*Intramyelinic edema		X
		Vacuolation		X
	Vascular
		Cholesterol clefts			X
		Hemorrhage		X
		*Hydrocephalus		X
		Hydromyelia			X
		Infarct		X
		Infiltrate, inflammatory cell		X
		Inflammation		X
		Mineralization			X
		Pigment		X
		Syringomyelia			X
		Syringomyelia/hydromyelia			X
Thrombus		X
Proliferative/non neoplastic
	Aggregates, granular cell				X
Proliferative/neoplastic
	*Medulloblastoma (M)			X
	Neuromyoblastoma (M)				X
	*Astrocytoma (M)			X
	Glioma, mixed (M)				X
	Oligodendroglioma (M)				X
	*Primitive neuroectodermal tumor (M)			X
	*Peripheral nerve sheath tumor, benign (B)			X
	*Peripheral nerve sheath tumor, malignant (M)			X
	^Tumor, granular cell (B)				X
	Tumor, granular cell (M)				X
	Meningioangiomatosis				X
	Meningioma, benign (B)			X
	Meningioma, malignant (M)			X
	Ependymoma (B)				X
	*Ependymoma (M)			X
	*Neuroblastoma (M)			X
	Papilloma, choroid plexus				X
	Carcinoma, choroid plexus				X
	Reticulosis, malignant				X
	Common artifacts
		Dark neuron artifact		X
		Myelin bubbles		X
		*Vacuolation, white matter		X

* Terminology with diagnostic criteria or comments described in the main text.

^ Example of benign granular cell tumor in chapter 3, fish digestive system, Figs. 3.23 and 3.24.

Ectopic tissue, brain, spinal cord and nerves – Similar to rodent

(Fig. 8.1)

Fig. 8.1.

Japanese medaka. Brain. Ectopic tissue, male germ cells (spermatogenesis), meninges. H&E.

Comments

Ectopic tissues have been observed in the central nervous system of fish to include gonadal tissue in the meninges of brain (Fig. 8.1).

Brain, Spinal Cord and Nerves – Non-Proliferative

Neuron-cell body

Cellularity decreased, neuron – Similar to rodent

Comments

This finding must be distinguished from artifactual changes. Neural tissue is often loosely arranged and it can be challenging to differentiate artifact due to suboptimal fixation from true decreases in cellularity. Regional variations in neuronal numbers should be compared to controls in similar sections with respect to age, species and stage of fish development.

Neuron necrosis/apoptosis due to experimental genetic mutations have been documented^{19, 22}. Regeneration of adult zebrafish neurons has been documented which is not typically observed in rodents or humans^{23, 24}. When there is a decrease of neuron numbers without accompanying glial cell response in a developmental toxicity study, neuron hypoplasia may be the preferred term as in rodents²⁵.

Fungus, brain – Similar to rodent

(Figs. 8.2, 8.3, 8.4 and 8.5)

Fig. 8.2.

Zebrafish. Peripheral nerve. Fungus, intracellular (microsporidian; presumptive Pseudoloma neurophilia). H&E. Image courtesy of Jeffrey Wolf.

Fig. 8.3.

Zebrafish. Brain, brainstem. Fungus, intracellular (Pseudoloma spp.). H&E. Image courtesy of Jeffrey Wolf.

Fig. 8.4.

Zebrafish. Brain. Fungus, intracellular (Pseudoloma spp.). H&E.

Fig. 8.5.

Zebrafish. Spinal cord. Fungus (Phaeohyphomycosis). H&E. Image courtesy of Laura Baseler.

Other Terms

• Phaeohyphomycosis, Chromomycosis, Microsporidia, Microsporidiosis

Modifier/Qualifier

• Pseudoloma species

• Microsporidia

Pathogenesis

• Opportunistic hyphal fungi (e.g. Cladosporium spp. or Exophiala spp.) via food or water, with routes of entry including cutaneous wounds or abrasions, the gills or via oral ingestion; predisposing factors include mutant genetic background, stress, water quality issues, concurrent trauma or disease

• Microsporidia: Transmission occurs via the ingestion of the infective spore stage either from the water or from cannibalized fish tissues. They have been detected in other tissues like ovaries and developing follicles indicating the possibility of vertical transmission or transmission through sexual products released during spawning

Diagnostic Features

• Primary predilection site is the central nervous system (spinal cord and hindbrain) and nerve roots. The parasite is also detected in skeletal muscle, kidney, ovaries and occasionally spine²⁶

• Xenomas present primarily in the CNS, measure up to 200 µm is size and contain aggregates of uninucleate spores segregated within sporophorous vesicles. Although rare, the sporoblasts and presporoblast stages are found randomly distributed throughout the xenoma

• Special stains like Luna stain bind to chitin and stains spores either bright red or orange, or Fungi-Fluor that fluorescently labels spores, could be utilized for diagnosis. In addition, Gram, Fites acid-fast, and Giemsa are variably effective in staining spores

• PCR based detection tests are also developed for Pseudoloma neurophilia²⁷

Comments

While a few examples of potentially pathogenic fungal organisms, including the microsporidia Pseudoloma neurophilia, have been provided above as modifiers/qualifiers, many more have been observed and/or reported in fish^{28, 29}.

Necrosis, neuron – Similar to rodent

Comments

Neurons are especially vulnerable to hypoxia and cell death due to toxins, ATP deletion and bacterial or viral infections³⁰. Focal necrosis involves the softening or loss of brain tissue after cerebral ischemia, infarction, infection, nutritional (vitamin E) imbalance, craniocerebral trauma, or other injury³¹.

Neuronophagia – Similar to rodent

Comments

In toxicant induced neuronophagia, infiltrating cells are not as prominent compared to immune or viral mediated diseases. Dying neurons are surrounded by microglia due to altered cell surface markers in degenerating or infected neurons or due proinflammatory cytokines and altered cell surface markers in degenerating neurons^{32, 33}.

Vacuolation, neuron – Similar to rodent

Comments

Vacuolation typically occurs in white matter of brain and myelinated peripheral nerves but may also be seen in neuronal bodies and glial cells. Neuron vacuolation should be carefully distinguished from artifact due to improper fixation and handling (especially prolonged ethanol immersion during dehydration), but routine processing procedures that employ ethanol would not be expected to induce artifactual vacuolation.

In fish, neuron vacuolation has been reported due to heavy metal toxicity, ultraviolet induced toxicity and infectious diseases^{34,35,36,37,38}.

Neuron-axon

Atrophy, axon – Similar to rodent

Comments

In zebrafish, axonal atrophy is mainly demonstrated in several human disease models by deletion of genes (e.g. smn, nrx2, tdp 43, and cldn) involved in various cellular process^39,40,41.

Degeneration, axon – Similar to rodent

Comments

In contrast to mammals, fish and amphibia functionally regenerate axons in the central nervous system⁴². Usually in fish, attenuation or absence of the myelin sheaths are noted while containing the cellular debris that are accompanied by inflammatory cells in late stages.

Glia-cell body

Gliosis – Similar to rodent

(Figs. 8.6 and 8.7)

Fig. 8.6.

Atlantic salmon. Brain. Gliosis, focal. H&E, 200×. Image courtesy of Jeffrey Wolf.

Fig. 8.7.

Lionfish. Brain. Gliosis and satellitosis. H&E, 400×. Image courtesy of Jeffrey Wolf.

Microgliosis – Similar to rodent

Comments

In chronic lesions, these cells transform from elongate spindle shaped to round cells with cellular debris and termed as gitter cells^{8, 11, 43}.

Satellitosis

(Fig. 8.7)

Other Terms

• Reactive oligodendroglia

Pathogenesis

• Response of oligodendroglia to degenerating neurons

Diagnostic Features

• Accumulation of oligodendroglia around the degenerating neuron cell body³²

Glia-myelin

Intramyelinic edema – Similar to rodent

Comments

Cytotoxic injury to either the myelin or the myelin producing cells leading to fluid accumulation between myelin layers. It is most commonly associated with lipophilic compounds like hexachlorophene and triethyltin^44,45. Due to the loose arrangement of white matter in fish, care must be taken by the pathologist to differentiate this finding from artifact in the central nervous system.

Vascular

Hydrocephalus – Similar to rodent

Comments

This finding is often observed with TCDD toxicity⁴⁶.

Brain, Spinal Cord and Nerves – Proliferative Neoplastic

Neural neoplasms may occur spontaneously in older adult fishes. In addition, neoplasms are encountered when fish species like zebrafish are frequently used as cancer model due to ease of tumor induction and the ability to image tumor model progression in living fish. Zebrafish share high genetic homology with humans, making them an attractive model for human neoplasia. Despite these similarities, there may be lower tumor incidence and differences in tumor spectra when compared to rodents and humans^{14, 47}. For example in zebrafish, most spontaneous or induced primary neurogenic neoplasms of the central nervous system (CNS) are poorly differentiated, highly embryonal neuroblastomas or primitive neuroectodermal tumors (PNETs). Some of these differences are attributable due to the abundant embryonal periventricular cells of the forebrain, diencephalon and myelencephalon^{14, 48}. Another notable difference is that mutations in tumor suppressor genes result in unusually high incidence of nerve sheath neoplasia (cranial and peripheral) in zebrafish compared to mammalian species, where a greater variety of neoplasia develops.

Medulloblastoma (M)

(Figs. 8.8 and 8.9)

Fig. 8.8.

Zebrafish, brca2 homozygous null. Brain. Medulloblastoma. H&E, 200×.

Fig. 8.9.

Zebrafish, brca2 homozygous null. Brain. Medulloblastoma (higher magnification of Fig. 8.8). H&E.

Diagnostic Features

• Highly cellular mass comprised of neuroepithelial stem cells exhibiting mainly neuronal differentiation

• Formation of whorls and pseudorosettes (i.e., concentric arrangement of tumor cells encircling small blood vessels) is common; pseudorosettes may have central fibrillary material

• Invasive growth pattern

Comments

Member of the Primitive Neuroectodermal Tumor (PNET) family of tumors derived from cells of cerebellum in mammals; however, in fish most of these tumors are observed in the midbrain.

Astrocytoma (M) – Similar to rodent (B, M)

(Figs. 8.10, 8.11, and 8.12)

Fig. 8.10.

Zebrafish, ras mutant. Brain. Glioblastoma. Gross image.

Fig. 8.11.

Zebrafish, ras mutant. Brain. Glioblastoma. H&E, 25×.

Fig. 8.12.

Zebrafish, ras mutant. Brain. Glioblastoma. H&E.

Comments

Gliomas, including astrocytomas, are rarely observed in fish, unless they are zebrafish genetically manipulated to be tumor models. In rodents and humans, these tumors are further classified to various subtypes based on histological features. In fish species, many of these subtype classifications are not reported due to their low incidence. Trangenic zebrafish expressing mutant ras genes rapidly develop gliomas including glioblastoma⁴⁹.

Primitive neuroectodermal tumor – Similar to rodent

(Fig. 8.13)

Fig. 8.13.

Zebrafish. Head. Primitive neuroectodermal tumor invading and replacing most normal brain tissue. H&E.

Peripheral nerve sheath tumor (M)

(Fig. 8.14)

Fig. 8.14.

Goldfish. Skin. Peripheral nerve sheath tumor, malignant. Note the typical storiform pattern that also characterizes the appearance of such tumors in mammals. H&E, 100×. Source: RTLA1643 (See introduction for RTLA explanation). Image courtesy of Jeffrey Wolf.

Comments

Goldfish, zebrafish, and certain other fish species are predisposed to develop neoplasia of the nerve sheath of peripheral and cranial nerves. Inactivating mutations in tumor suppressor genes (tp53, mlh1, msh2, msh6, and ribosomal genes) results in induction¹⁴.

A differential diagnosis for this tumor is a syndrome described in the bicolor damselfish which has gained interest as potential model for neurofibromatosis 1 in humans, and appears grossly as multicentric tracking along cutaneous nerves on the body and fins, as well as spinal nerves. Histologically there are invasive proliferations of spindle cells in various patterns, consistently associated with peripheral nerves⁵⁰. A virus-like agent has been postulated for this transmissible neoplastic disorder.⁵¹

Malignant ependymoma (M)

(Figs. 8.15 and 8.16)

Fig. 8.15.

Zebrafish. Spinal cord. Malignant ependymoma. H&E.

Fig. 8.16.

Zebrafish. Spinal cord. Malignant ependymoma (higher magnification of Fig. 8.15). H&E.

Other Terms

• Ependymoblastoma

Diagnostic Features

• The tumor cells are polygonal, fusiform, or triangular with delicate pinpoint chromatin

• A characteristic feature of malignant ependymoma is that the tumor cells are arranged in epithelium-like layers, lining a central cyst⁵²

Neuroblastoma, malignant

(Figs. 8.17 and 8.18)

Fig. 8.17.

Zebrafish. Brain. Neuroblastoma following embryonal administration of methylazoxymethanol acetate (MAMA). H&E.

Fig. 8.18.

Zebrafish. Brain. Neuroblastoma following embryonal administration of methylazoxymethanol acetate (MAMA) (higher magnification of Fig. 8.17). H&E.

Diagnostic Features

• Neuroblasts in trabecular patterns interspersed by glial fibrillar material and linear cavities resembling central neural canals lined by ependyma-like cells⁵³

• Tumor cells have high mitotic index, are small with indiscernible cytoplasm and contain vague cytoplasmic borders

Brain, Spinal Cord and Nerves – Common Artifacts

Vacuolation, white matter

Comments

White matter of the nervous system has high lipid content compared to other organs. Common histological processing results in dissolution of lipid present in the nervous system, and often results in the formation of fine vacuoles. This situation is further complicated in fish species where the white matter is more loosely organized. Autolysis and prolonged fixation increases artifactual vacuolation, which is usually not accompanied by other histological features associated with typical vacuolar changes.