Argyrophilic dark neurons represent various states of neuronal damage in brain insults: some come to die and others survive

doi:10.1016/j.neuroscience.2004.02.002

Neuroscience

Volume 125, Issue 3, 2004, Pages 633-644

https://doi.org/10.1016/j.neuroscience.2004.02.002 Get rights and content

Abstract

Argyrophilic dark neurons (DNs) reflect the early histopathological state of neurons following various brain insults. We examined the fate of DNs, about to either die or recover, following two types (heavy and light damage) of brain insult. Wistar rats were injected ibotenic acid unilaterally into the hippocampal CA1 region (ibotenic acid [IA] injection) or were forced to swim (SWIM). Argyrophil III (DNs)-, activated caspase-3 immuno-, TUNEL- and hematoxylin–eosin (H-E)-staining and ultrastructural examinations were then performed.

One to three hours after IA injection, typical DNs (argyrophilic both in somata and dendrites) with corkscrew-like dendrites were densely packed in the pyramidal cell layer of hippocampal CA1 around the injection site. After 12–24 h, DNs were argyrophilic only in the somata and proximal dendrites but absent in distal dendrites in the CA1 region. However, at this time typical DNs were found in remote areas. At 3 h, caspase-3 activation was detected at the injection site, which increased to a peak level after 12 h. Three to 7 days after injection, TUNEL positive cells were detected in the CA1 pyramidal cell layer. Immediately following SWIM, “brown” rather than “dark” neurons were detected in the various areas and most frequently in the CA1 pyramidal cell layer. No typical DNs were detected over the first 3 days. Some activation of caspase-3 was detected in a few CA3 pyramidal cells but no TUNEL-positive cells were detected.

Ultrastructural examination revealed a diffuse distribution of aggregated silver particles in the dendrites and cytoplasm of pyramidal cells at the sites of IA injection. After SWIM, silver particles were detected mainly on mitochondria of affected cells.

These data suggest that DNs provide a measure of neuronal damage: typically dark neurons with broad damage to the cytoskeleton of dendrites would die, while non-typical brown neurons, that may have a disturbance in mitochondria, predominantly survive.

Section snippets

Animals

Adult male Wistar rats weighing 250–280 g were used. They were kept in a temperature-controlled room at 23–25 °C with free access to standard laboratory food and water. Animal care was in accordance with the “Guidelines of the Care and Use of Laboratory Animals” and approved by the Committee of The Institute of Experimental Animals, Nagoya City University Graduate School of Medical Sciences and with the NIH Guide for the Care and Use of Laboratory Animals. All efforts were made to minimise the

Argyrophilic DNs following IA injection into the hippocampus

One hour after the injection of IA into the hippocampus, argyrophilic DNs were detected in the pyramidal cell layer of the CA1 region at the site of injection (Fig. 1A). Three hours after injection, abundant dense-argyrophilic DNs were detected in the same area (Fig. 1C). The pyramidal neurons were argyrophilic in somata and dendrites possessed of corkscrew-like dendrites (Fig. 1D). Using H-E staining, pycnotic cells were detected in the same CA1 area where DNs were abundant (Fig. 1E). Twelve

Discussion

Argyrophil III staining is a useful method for detecting DNs, and provides a measure of the early histopathological state of neurons after various brain insults, such as brain ischemia (Czurko and Nishino, 1993, Hsu et al., 1994, Cizkova et al., 1996, Onizuka et al., 1996), neurodegenerative disease (Chui et al., 1999), head injury (Van Den Pol and Gallyas, 1990, Gallyas and Zoltay, 1992a, Gallyas et al., 1992b), ES (Gallyas et al., 1993; Ishida et al., 1997), and intoxication with neurotoxin (

Acknowledgements

K.I. and H.N. were supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan, Scientific Research No. 15500360 and No. 15200026, and H.N. was supported by Special Coordination Funds, No. 13073-2125-14.

References (24)

B Ahlemeyer et al.
Release of cytochrome c into the extracellular space contributes to neuronal apoptosis induced by staurosporine
Brain Res
(2002)
A Czurko et al.
‘Collapsed’ (argyrophilic, dark) neurons in rat model of transient focal cerebral ischemia
Neurosci Lett
(1993)
C.J Faherty et al.
Caspase-3-dependent neuronal death in the hippocampus following kainic acid treatment
Mol Brain Res
(1999)
K Inoue et al.
Microtubule-associated protein 2 expressing COS7 cells are resistant to argyrophilia under oxygen- and glucose-free condition
Exp Neurol
(1996)
K Ishida et al.
Argyrophilic dark neurons distribute with a different pattern in the brain after over hours treadmill running and swimming in the rat
Neurosci Lett
(1999)
K Mizoguchi et al.
Stress induces neuronal death in the hippocampus of castrated rats
Neurosci Lett
(1992)
K Onizuka et al.
Early cytopathic features in rat ischemia model and reconstruction by neural graft
Exp Neurol
(1996)
R Siman et al.
Immunolocalization of caspase proteolysis in situEvidence for widespread caspase-mediated apoptosis of neurons and glia in the postnatal rat brain
Neuroscience
(1999)
M Tanaka et al.
In situ detection of activated caspase-3 in apoptotic granule neurons in the developing cerebellum in slice cultures and in vivo
Dev Brain Res
(2000)
T Yoshimoto et al.
NXY-059 maintains Akt activation and inhibits release of cytochrome C after focal cerebral ischemia
Brain Res
(2002)

D Cizkova et al.

Ischemic damage in the hippocampusA silver impregnation and immunocytochemical study in the rat

Arch Italiennes Biol

(1996)

D.H Chui et al.

Transgenic mice with Alzheimer presenilin 1 mutation without amyloid plaque formaton

Nat Med

(1999)

Cited by (42)

Chronic exposure to methadone impairs memory, induces microgliosis, astrogliosis and neuroinflammation in the hippocampus of adult male rats
2022, Journal of Chemical Neuroanatomy
Methadone is a centrally-acting synthetic opioid analgesic widely used in methadone maintenance therapy (MMT) programs throughout the world. Given its neurotoxic effects, particularly on the hippocampus, this study aims to address the behavioral and histological alterations in the hippocampus associated with methadone administration. To do so, twenty-four adult male albino rats were randomized into two groups, methadone treatment and control. Methadone was administered subcutaneously (2.5–10 mg/kg) once a day for two consecutive weeks. A comparison was drawn with behavioral and structural changes recorded in the control group. The results showed that methadone administration interrupted spatial learning and memory function. Accordingly, treating rats with methadone not only induced cell death but also prompted the actuation of microgliosis, astrogliosis, and apoptotic biomarkers. Furthermore, the results demonstrated that treating rats with methadone decreased the complexity of astrocyte processes and the complexity of microglia processes. These findings suggest that methadone altered the special distribution of neurons. Also, a substantial increase was observed in the expression of TNF-α due to methadone. According to the findings, methadone administration exerts a neurodegenerative effect on the hippocampus via dysregulation of microgliosis, astrogliosis, apoptosis, and neuro-inflammation.
Evaluation of Fluoro-Jade C Staining: Specificity and Application to Damaged Immature Neuronal Cells in the Normal and Injured Mouse Brain
2020, Neuroscience
Fluoro-Jade C (FJC) staining is widely used for the specific detection of all degenerating mature neurons, including apoptotic, necrotic, and autophagic cells. However, whether FJC staining can detect degenerating immature neurons and neural stem/precursor cells remains unclear. In addition, some conflicting studies have shown that FJC and its ancestral dyes, Fluoro-Jade (FJ) and FJB, can label resting/activated astrocytes and microglia. In the present study, we examined the validity of FJC staining for the detection of neuronal cells in adult and embryonic mouse brains under normal and injured conditions. In the adult rodent subventricular zone (SVZ)–rostral migratory stream (RMS)–olfactory bulb (OB) system, apoptosis associated with neurogenesis occurs under normal conditions. Using this system, we detected FCJ positive (+) cells, some of which were doublecortin (DCX)(+) neuroblasts, in addition to neuronal nuclei (NeuN)(+) mature neurons. FJC negative (−) apoptotic cells expressing activated Caspase 3 were also observed, and a small number of FJC(+)/ionized calcium-binding adaptor molecule 1 (Iba1)(+) microglia and FJC(+)/glial fibrillary acidic protein (GFAP)(+) astrocytes were observed in the normal brain. Next, we analyzed embryonic brains, in which the apoptosis of neural stem/precursor cells was induced by the administration of N-ethyl-N-nitrosourea (ENU) or ethanol at embryonic day 14 or 10, respectively. In those brains, FJC(+) neural stem/precursor cells and neuroepithelial cells expressing SRY-related HMG-box 2 (Sox2) were observed. Surprisingly degenerating mesenchymal cells were also FJC(+). The present study indicates that FJC is a reliable marker for degenerating neuronal cells during all differentiation stages. However, FJC could also label degenerating non-neuronal cells under some conditions.
Progesterone modulates post-traumatic epileptogenesis through regulation of BDNF-TrkB signaling and cell survival-related pathways in the rat hippocampus
2019, Neuroscience Letters
Female sex hormone, progesterone, in addition to seizure modifying activity is also known as a potential protective agent against various brain injury conditions. Considering the predisposal role of traumatic brain injury (TBI) on developing post-traumatic epilepsy (PTE), the effect of progesterone on post-traumatic epileptogenesis is not investigated yet. Male Wistar rats were given a moderate focal weight drop injury (500 gr) or sham surgery and then progesterone (16 and 32mg/kg) was given daily for two consecutive weeks. On day 15 of injury, seizures were induced by administration of a GABAA receptor antagonist, pentylenetetrazole (PTZ, 30 mg/kg). Seizures were then assessed over a 1-h period using the Racine clinical rating scale.
Traumatized animals that received 32 mg/kg progesterone had reduced score, duration of seizures and almost did not show tonic-clonic seizures during 60 min versus the untreated trauma group. In line with behavioral alterations, 32 mg/kg progesterone enhanced the amount of Nrf2 and HO-1 proteins and decreased the level of NF-kB, BDNF, Caspase 3 and ratio of Bax/Bcl-2 in the ipsilateral hippocampus. Additionally, the number of TUNEL-positive apoptotic cells, as well as injured dark neurons in the parietal cortex and hippocampal CA1 of 32 mg/kg-treated animals showed a significant reduction. Administration of 16 mg/kg progesterone elevated production of BDNF, Bax and Caspase 3 and decreased anti-apoptotic Bcl-2 protein. Taken together, an early administration of 32 mg/kg of progesterone after TBI for two weeks post-injury modified seizure activity.
Our findings suggest that post-traumatic anti-epileptogenesis property of a high dose of progesterone partly occurs through the manipulation of BDNF-TrkB axis along with control of cell survival pathways.
Vitamin C prevents hypothyroidism associated neuronal damage in the hippocampus of neonatal and juvenile rats: A stereological study
2018, Journal of Chemical Neuroanatomy
Hypothyroidism causes an imbalance in antioxidant and pro-oxidants criteria in the brain and enhances the concentration of reactive oxygen species (ROS), and neuronal damage has been observed following an excessive ROS. The main purpose of this study was to examine the preventive effect of vitamin C on hypothyroidism associated neuronal damage in the hippocampus of neonatal and juvenile rats.
Pregnant rats after delivery of their pups were randomly divided into four groups and treated with (1) normal drinking water as a control group, (2) Propylthiouracil (PTU) 0.005% added to drinking water, (3-, 4) PTU + Vit C 10 mg/ kg and PTU + Vit C 100 mg/ kg to drinking water.
Treatment was carried out during rat’s lactation period until to the postnatal day (PND) 60. To assess the histological and stereological changes that occur in this study, brains of 5 male pups were extracted.
The number of dark neurons and apoptotic cells in the hippocampal sub-regions of PTU group was significantly greater than the control group’s hippocampal sub-regions. In addition, hypothyroidism induced a reduction in the hippocampal volume and increased the numerical density and the total amount of dark neurons. The vitamin C only dose of 100 mg/kg significantly reduced the number of dark neurons and apoptotic cells (P < 0.01) and considerably weakened the influence of hypothyroidism on the volume reduction of the hippocampus (P < 0.05).
The current study suggested that vitamin C administration has a possibility to prevent hippocampal neuronal damage caused by neonatal and juvenile hypothyroidism in rats.
The effects of Nigella sativa on neural damage after pentylenetetrazole induced seizures in rats
2016, Journal of Traditional and Complementary Medicine
Nigella sativa (NS) has been suggested to have neuroprotective and anti-seizures properties. The aim of current study was to investigate the effects of NS hydro-alcoholic extract on neural damage after pentylenetetrazole (PTZ) – induced repeated seizures. The rats were divided into five groups: (1) control (saline), (2) PTZ (50 mg/kg, i.p.), (3–5) PTZ-NS 100, PTZ-NS 200 and PTZ-NS 400 (100, 200 and 400 mg/kg of NS extract respectively, 30 min prior to each PTZ injection on 5 consecutive days). The passive avoidance (PA) test was done and the brains were then removed for histological measurements. The PTZ-NS 100, PTZ-NS 200 and PTZ-NS 400 groups had lower seizure scores than PTZ group (P < 0.01 and P < 0.001). The latency to enter the dark compartment by the animals of PTZ group was lower than control in PA test (P < 0.01). Pre-treatment by 400 mg/kg of the extract increased the latency to enter the dark compartment (P < 0.05). Meanwhile, different doses of the extract inhibited production of dark neurons in different regions of hippocampus (P < 0.001). The present study allows us to suggest that the NS possesses a potential ability to prevent hippocampal neural damage which is accompanied with improving effects on memory.
Influence of metformin on mitochondrial subproteome in the brain of apoE knockout mice
2016, European Journal of Pharmacology
Citation Excerpt :
It is tempting to speculate, that metformin may reinforce the removal of dysfunctional cells. According to Ishida et al. (2004) mitochondria are the key switching point of the life and death: population of neurons with disturbances in mitochondria predominantly would survive (non-typical “brown” neurons), while those with broad damage to the cytoskeleton would undergo apoptosis (“dark” neurons). Clearly, the involvement of apoptotic mechanisms in metformin action in apoE−/− mice requires further elucidation.
Neurodegenerative diseases are the set of progressive, age-related brain disorders, characterized by an excessive accumulation of mutant proteins in the certain regions of the brain. Such changes, collectively identified as causal factors of neurodegeneration, all impact mitochondria, imminently leading to their dysfunction. These observations predestine mitochondria as an attractive drug target for counteracting degenerative brain damage.
The aim of this study was to use a differential proteomic approach to comprehensively assess the changes in mitochondrial protein expression in the brain of apoE-knockout mice (apoE^−/−) and to investigate the influence of prolonged treatment with metformin – an indirect activator of AMP-activated protein kinase (AMPK) on the brain mitoproteome in apoE^−/− mice.
The quantitative assessment of the brain mitoproteome in apoE^−/− revealed the changes in 10 proteins expression as compared to healthy C57BL/6J mice and 25 proteins expression in metformin-treated apoE^−/− mice. Identified proteins mainly included apoptosis regulators, metabolic enzymes and structural proteins.
In summary, our study provided proteomic characteristics suggesting the decrease of antioxidant defense and structural disturbances in the brain mitochondria of apoE^−/− mice as compared to healthy controls. In this setting, the use of metformin changed the expression of several proteins primarily involved in metabolic processes, the regulation of apoptosis and the structural maintenance of mitochondria, what could potentially restore their native functionalities.

View all citing articles on Scopus

View full text

Argyrophilic dark neurons represent various states of neuronal damage in brain insults: some come to die and others survive

Abstract

Section snippets

Animals

Argyrophilic DNs following IA injection into the hippocampus

Discussion

Acknowledgements

Brain Res

Neurosci Lett

Mol Brain Res

Exp Neurol

Neurosci Lett

Neurosci Lett

Exp Neurol

Neuroscience

Dev Brain Res

Brain Res

Ischemic damage in the hippocampusA silver impregnation and immunocytochemical study in the rat

Arch Italiennes Biol

Transgenic mice with Alzheimer presenilin 1 mutation without amyloid plaque formaton

Nat Med