Brain Microanatomy: Artifacts, Neoplastic Lesions and Utilization of Special Stains

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2 Brain Microanatomy: Artifacts, Neoplastic Lesions and Utilization of Special Stains Current Standard for CNS Evaluation in Rodent Toxicity Studies ESTP Congress in Ghent, Belgium 12 September 2013 Robert H. Garman, DVM Consultants In Veterinary Pathology, Inc. Monroeville, PA

3 Neurocytology, Including Cell Types, Cell Reactions and Artifact Cells of Neuroectodermal Origin Neurons Astrocytes Oligodendrocytes Ependymocytes Cells of Mesenchymal Origin Microglia (as well as blood vessels, adipose tissue, and meninges) 3

4 Large Motor Neurons 4

5 Large Sensory Neurons 5

6 Mixtures of Large Neurons and Small Neurons (Granule Cells) 6

7 Olfactory Bulb Cochlear Nucleus 7

8 Normal Striatum Striatum (Hemorrhagic Shock) 8

9 Locations of all 7 circumventricular organs in a rat brain (LFB/CV stain) 9

10 6 CVOs from a rat brain a = organum vasculosum of lamina terminalis b = subfornical organ c = mediam eminence d = subcommissural organ e = pineal F = area postrema Garman RH (2011) Histology of the Central Nervous System. Toxicol Pathol, 39:

11 Necrotic neurons versus Artifactual Dark Neurons Red Dead (Eosinophilic) Neuron (left) Dark Neuron - representing a handling artifact - (right) 11

12 Dark neurons tend to cluster 12

13 Dark Neurons Are most often the result of handling. 13

14 Drying Artifact Saline Artifact 14

15 Dark neuron artifact is most apparent in large cytoplasm-rich neurons 15

16 In cresyl violetstained sections, dark neurons are not degenerative! 16

17 Dark Neurons (H&E) Eosinophilic Neurons (H&E) 17

18 Eosinophilic Neurons (H&E) Eosinophilic Neurons (Cresyl Violet) 18

19 Dark Neurons vs. Dead Neurons 1) Dark neurons are monomorphic; eosinophilic neurons are characterized by different stages of degeneration. (The only exception may shortly after a peracute injury.) 2) Eosinophilic neurons are accompanied by additional changes such as secondary cellular reactions and, occasionally, an altered neuropil. 19

20 Unilateral ischemic insult, rat brain 20

21 Eosinophilic neurons in different stages of degeneration 21

22 Swollen neuron, eosinophilic neurons, karyorrhexis and active microglial cell 22

23 Fluoro-Jade Stains for Degenerating Neurons 1) Lower the threshold (magnification) for detection. 2) Allow observation of degenerative neuronal processes as well as cell bodies. 3) Allow for the detection of small-sized degenerating neurons for which it is difficult to detect an eosinophilic cytoplasmic change. 4) Assist in the detection of peracute degenerative processes (prior to development of eosinophilia). 23

24 Retrosplenial cortex (rat) 48 hrs. after MK-801 treatment 24

25 Retrosplenial cortex (rat) 48 hrs. after MK- 801 treatment (H&E- vs. Fluoro- Jade B-stained sections) 25

26 Hippocampal CA1 sector (rat) 24 hrs. after fentanyl treatment (H&E- vs. Fluoro- Jade B-stained sections) 26

27 CA1 sector of a rat hippocampus 48 hours after a drug treatment. H&E vs. Fluoro-Jade B for detecting degeneration in smallsized interneurons. 27

28 Higher magnification images for degenerating small-sized interneurons. 20x 40x 28

29 Peracute injury (due to a one-hour grand mal seizure). 29

30 Adenosine A1 KO mouse killed after a one-hour grand mal seizure. H&E vs. Fluoro-Jade C 30

31 H&E Dark Neurons in H&E and Fluoro- Jade-stained sections Fluoro-Jade 31

32 Fluoro-Jade versus Silver Degeneration Stains 1. Frozen sections are necessary for silver degeneration stains. F-J stains may be used on frozen or paraffin sections. 2. Silver degeneration stains require greater technical skill/may have more artifactual staining. 3. Silver degeneration stains are archivable. (Fluoro- Jade will fade while viewed with fluorescent light, although the shelf life of stained slides is fairly long.) 4. Silver degeneration stains show greater neuronal process detail. 32

33 Fluoro-Jade B, retrosplenial cortex of a rat 48 hours after MK- 801 treatment. Section from the same rat and the same (contralateral) brain level stained with amino cupric silver. 33

34 Unilateral Optic Nerve Atrophy in a rat (H&E, F-J B and cupric silver) 34

35 Different levels of midbrain (and different time points) showing axonal injury with retraction ball formation. Garman RH et al. (2011) Blast exposure in rats with body shielding is characterized primarily by diffuse axonal injury. J Neurotrauma, 28(6)

36 For Axonal Injury Silver degeneration stains (e.g. amino cupric silver) are the stains of choice if you can use frozen sections. Amyloid precursor protein (APP) stains are O.K. for peracute axonal injury if you are limited to doing IH on paraffin sections. Non-degenerative silver stains such as Bodian s, Bielschowsky s, Gallyas are also good for axonal injury. 36

37 Non-degenerative silver stain for internal neuronal structure (Bielschowsky s) 37

38 Astrocytes 1. Provide nutritional support of neurons and maintenance of extracellular electrolyte & neurotransmitter levels. Crucial for neuron function and survival. 2. Signal other astrocytes via gap junctions. 3. Have neurotransmitter receptors similar to those of neurons. 4. Are an important component of the BBB & also regulate blood flow. 5. Principle marker = GFAP. 38

39 Normal-appearing astrocytes, H&Estained section of cerebral cortex. 39

40 Examples of reactive and gemistocytic cytoplasm-rich astrocytes. 40

41 Normal GFAP-stained astrocytes ( star cells ), cerebral cortex.41

42 Reactive astrocytes have thickened cytoplasmic processes. 42

43 Hippocampal sclerosis, CA1 sector H&E Hippocampal sclerosis, CA1 sector (GFAP) 43

44 Multiple brain sections from a clinically normal rat!!! 44

45 Vacuoles: Artifact or a Peracute Alteration? 45

46 Cerebellar Vacuoles; H&E, GFAP and Fluoro-Jade B. 46

47 One Example of Good Artifact Dog cerebral cortex, H&E stain. Alzheimer type II astrocyte change due to experimentallyinduced hyperammonemia. Images courtesy of Dr. Michael D. Norrenberg 47

48 Are these changes also examples of good artifact? 48

49 Two Types of Brain Edema Francesca & Rezzani (2010). Aquaporin and blood brain barrier Curr Neuropharm 8:

50 GFAP (Green) vs. Aquaporin 4 (Red) Simard M et al (2003). Signaling at the gliovascular interface, JNS, 23(27): ,

51 Astrocytes and Aquaporins in the Pathogenesis of Cytotoxic Brain Edema and/or in Postmortem Swelling of Astrocytes 1) Astrocyte nuclear or cytoplasmic swelling may occur in certain metabolic diseases. 2) Astrocyte swelling (especially cytoplasmic) also represents a very common postmortem artifact. 3) This swelling may be enhanced by certain pharmacologic agents. 51

52 Oligodendrocytes 1. Myelin-forming cells of the CNS. 2. One oligodendrocyte myelinates multiple axons (as many as 50 or more). 3. Para-neuronal oligodendrocytes are often referred to as satellite cells. 52

53 Left: Classic Artifactual Fried Egg Appearance of Oligodendrocytes Right: Normal Oligodendrocytes in a Perfusion-fixed Brain 53

54 Left side micrograph: Within the brain s fiber tracts, oligodendrocytes are typically arranged in rows. Right side micrograph: Some cranial nerves show an extra-axial transition between a central (right) to a peripheral (left) pattern of myelination. 54

55 Examples of Neoplastic Satellitosis 55

56 Microglia 1. Are the inflammatory cells of the CNS comparable to the blood monocytes and tissue histiocytes. 2. Constitute 5 20% of the brain cells. 3. Function in both pro-inflammatory ( classical activation and reparative ( alternative activation ) modalities. 4. Ionized calcium binding adaptor molecule 1 (Iba1) is often used to stain reactive microglia. 56

57 Find the single microglial cell in this section of normal cortex. H&E 57

58 Neuronophagia by activated microglial cells 58

59 Progression of neuronophagia by microglial cells 59

60 Microglia reacting to necrotic neurons CA1 sector of hippocampus 60

61 Iba1 Stain, cerebral cortex Reactive microglial cell, cerebral cortex 61

62 Ventricular fibrillation/cardiac arrest model, rat (14-days prior to death) H&E Iba1 62

63 Principal Histologic Artifacts in Brain Sections 1) Dark Neurons 2) Vacuolation of neurons, neuropil or myelin 63

64 Good (left) versus suboptimal preservation of DRG by perfusion fixation 64

65 Prominent vacuoles found sporadically in sensory ganglia of rats 65

66 Buscaino bodies ( Mucocytes ) An artifact of myelin. H&E 66

67 Contrasting Two Patterns of Intramyelinic edema 67

68 Buscaino bodies are occasionally refractile under polarized light. 68

69 Brain Microanatomy: Neoplastic Glial Lesions in Rodents Current Standard for CNS Evaluation in Rodent Toxicity Studies ESTP Congress in Ghent, Belgium 12 September 2013 Important Reference: Kaufmann, W et al (2012): Proliferative and non-proliferative lesions of the rat and mouse central and peripheral nervous system Toxicol Pathol. 40:

70 Classification of Proliferative Lesions Neuronal Medulloblastoma Neuromyoblastoma Glial/Schwann Cell Astrocytoma Glioma Oligodendroglioma Schwannoma Choroid plexus Papilloma Carcinoma Ependyma Ependymoma Meninges Granular cell aggregates Granular cell tumour Meningioangiomatosis Meningioma Other cell lineages Lipomatous hamartoma Malignant reticulosis Lymphoma 70

71 Historical Control Data* for Brain from the NTP F344 RATS Brain Tumor Incidence (M) Incidence (F) ASTROCYTOMA, High grade (aka. Malignant) 3/1248 0/1200 EPENDYMOMA, benign 1/1248 0/1200 EPENDYMOMA, malignant 0/1248 1/1200 GLIOMA**, low grade (aka. Benign) 0/1248 1/1200 GLIOMA**, high grade (aka. Malignant) 0/1248 2/1200 OLIGODENDROGLIOMA, low grade (aka. Benign) 1/1248 0/1200 OLIGODENDROGLIOMA, high grade (aka. Malignant) 1/1248 3/1200 GRANULAR CELL TUMOUR, BENIGN 2/1248 0/1200 SCHWANNOMA, MALIGNANT 1/1248 0/1200 Publically available at ** Has been used for glioma, mixed or astrocytoma 71

72 Historical Control Data* for Brain from the NTP B6C3F1 MICE Brain Tumour Incidence (M) Incidence (F) MENINGIOMA, benign 1/1150 0/1197 MENINGIOMA, malignant 1/1150 0/1197 OLIGODENDROGLIOMA, high grade (aka. Mal) 1/1150 0/1197 SCHWANNOMA, malignant 0/1150 2/1197 * Publically available at 72

73 Glial Cell Neoplasms in Rodents 1) Astrocytomas are the most common glial tumor in rats, followed by oligodendroglioma and mixed glioma 2) Primarily seen in older-aged rats (15 months +) 3) Immunohistochemistry suggests a histiocytic cell lineage for astrocytomas in rats, so the new terms that have been recommended are malignant microglial tumor or microglioma. 73

74 Astrocytoma: malignant, high grade Photo courtesy of Dr. James Morrison. 74

75 Astrocytoma: H&E, protoplasmic morphology Photo courtesy of Dr. James Morrison. 75

76 Astrocytoma: fibrillary morphology Photo courtesy of Dr. James Morrison. 76

77 Astrocytoma: neuronal satellitosis Photo courtesy of Dr. James Morrison. 77

78 Astrocytoma: extension into meninges Photo courtesy of Dr. James Morrison. 78

79 Astrocytoma: necrosis and palisading Photo courtesy of Dr. James Morrison. 79

80 Astrocytoma: cellular atypia Photo courtesy of Dr. James Morrison. 80

81 Oligodendroglioma: Photo courtesy of the NTP 81

82 Oligodendroglioma: Photo courtesy of the NTP 82

83 Oligodendroglioma: Photo courtesy of the NTP 83

84 Oligodendroglioma: Photo courtesy of the NTP 84

85 Oligodendroglioma: Photo courtesy of the NTP 85

86 Mixed Glioma: Photos courtesy of the NTP 86

87 Mixed Glioma: Photo courtesy of the NTP 87

88 Mixed Glioma: Photo courtesy of the NTP 88

89 Immunohistochemical Staining of Rat Glial Cell Neoplasms 1) Astrocytoma 2) Oligodendroglioma 3) Mixed gliomas (oligo + astrocytoma) 89

90 IHC on Rat Astrocytomas GFAP IHC Photos courtesy of Dr. James Morrison Iba-1 IHC 90

91 IHC on Rat Oligodendrogliomas Anti-Olig2 IHC Anti-GFAP IHC Images from: Kolenda-Roberts HM et al. (2013) Immunohistochemical characterization of spontaneous and acrylonitrile-induced brain tumors in the rat. Toxicol Pathol 41:98-108Pathology ` Copyright by Society of Toxicologic Pathology 91

92 IHC staining of mixed gliomas Staining for GFAP and for nestin (a marker of astrocyte precursors) Images from: Nagatani M et al (2013): GFAP-positive neoplastic astrocytes in spontaneous oligodendrogliomas and mixed gliomas of rats. Toxicol Pathol; 41:

93 Summary Comments Glial Neoplasms in Rats - Glial cell neoplasms are very uncommon in rats and are rare in mice; these neoplasms are primarily found in old rats. The most frequent glial cell neoplasm in the rat is the astrocytoma, but IHC has shown the tumor cells to stain for monocyte markers such as Iba1. The diagnostic terms microglioma or malignant microglial cell tumor may, therefore, be more appropriate. Oligodendrogliomas show IHC markers for oligodendrocytes. Some mixed gliomas have been shown to contain both neoplastic oligodendrocytes and GFAP + neoplastic astrocytes or nestin + cells. (Nestin is expressed in proliferating neuroepithelial cells, including astrocyte precursors.) 93

94 Neuropathology Overview 1. The brain is a highly heterogeneous organ. 2. Many neuroanatomic regions have unique sensitivities to physical or chemical insult. 3. Adequate evaluation for neurotoxic endpoints requires: a) Proper tissue preparation (to avoid artifact) b) Relatively extensive sampling c) Application of a variety of stains, when appropriate d) Pathologist s time and experience The regulatory agencies need to know which brain regions were examined.

95 Acquiring new scientific knowledge can be a pleasing experience. Un-retouched electron micrograph of normal mitochondria within a small myelinated axon. From : Haines, DE; Fundamental Neuroscience for Basic and Clinical Applications, 3 rd Ed. Elsevier, 2006

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