Methodological Approaches to Experimental Evaluation of Neuroprotective Action of Potential Drugs
Abstract
:1. Introduction
2. Preliminary Evaluation of the Neuroprotective Effects of Potential Drugs In Vitro
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- Intact: A suspension of neurons without the addition of initiating agents or potential neuroprotectors under investigation;
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- Control: A suspension of neurons to which agents that induce oxidative and nitrosative stress, glutamatergic excitotoxicity, and glutathione thiol–disulfide system deprivation are added at concentrations capable of causing the death of 50% of neurons (0.1–5 µM). To initiate oxidative stress in the neuron suspension, 0.25–1.0 mM H2O2 is added to the incubation medium. Glutamatergic “excitotoxicity” is induced by adding kainate (200–400 µM), glutamate (0.1–10 mM), or N-methyl-D-aspartate (100–150 µM) to the incubation medium. Glutathione thiol–disulfide system deprivation is achieved by introducing chloro-2,4-dinitrobenzene (CDNB) (50–500 µM), a selective inhibitor of glutathione S-transferase that forms conjugates with glutathione in cytosolic and mitochondrial fractions, into the incubation medium [16,17,18,19,20,21]. Alongside the intact and control samples, samples with the addition of initiating agents and pharmacological agents at various concentrations are prepared, followed by determining their effective concentration. The neuroprotective activity of potential neuroprotectors is assessed by counting neurons exhibiting signs of apoptosis using flow cytometry or histochemical methods. Our studies have shown that the addition of the aforementioned neurotoxins to the incubation medium led to a pronounced disruption of cellular, molecular–biochemical processes. These disruptions were consistent in nature but varied in degree of severity—accompanied by a sharp shift in the thiol–disulfide balance toward oxidized thiols (a significant decrease in reduced glutathione concentration and an increase in oxidized glutathione). An increase in the marker of oxidative protein damage—nitrotyrosine—was observed, along with a decrease in the activity of mitochondrial superoxide dismutase (Mn-SOD). We also recorded dynamic changes in the synthesis of endogenous cytoprotective factors—HSP and HIF proteins.
- MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced opening of mitochondrial pores: add 40–60 µM MPTP to the incubation medium and after 5 min, add 50 µM CaCl2;
- Ca2+-induced opening of mitochondrial pores: add 200 µM CaCl2 to the incubation medium;
- NO-induced opening of mitochondrial pores: add 20–100 µM sodium nitroprusside to the incubation medium and, after 2 min, add 50 µM CaCl2;
- H2O2-induced opening of mitochondrial pores: add 50 mM hydrogen peroxide to the incubation medium and, after 2 min, add 50 µM CaCl2.
3. Preliminary Assessment of the Neuroprotective Effects of Potential Pharmaceutical Agents Using Various Models of Cerebral Ischemia
4. Assessment of Neurological Deficit in Animals with Experimental Cerebral Ischemia Serves as an Integrative Measure of the Neuroprotective Efficacy of Potential Pharmacological Agents
- Mild: 0 to 3 points;
- Moderate: 3 to 7 points;
- Severe: 7 points and above.
- Unilateral partial ptosis: 0.5 points;
- Unilateral ptosis: 1 point;
- Tremor: 0.5 points;
- Circling movements: 0.5 points;
- Paresis of limbs (per limb): 1 point;
- Paralysis of limbs (per limb): 2 points;
- Lateral positioning: 3 points;
- Inability to remain on the rotating rod (3 RPM) for 4 min: 3 points.
5. Determination of Oxidative Stress Markers and Antioxidant System Status
- Pentafluorophenylhydrazine (PFPH);
- Methylhydrazine (MH);
- 4-(2-phthalimidyl)benzohydrazine (FBH);
- 2,4-Dinitrophenylhydrazine (DNPH);
- o-(2,3,4,5,6-Pentafluorobenzyl)hydroxylamine hydrochloride (PFBH);
- tert-Butyldimethylchlorosilane (BDMCS);
- N,O-Di-(trimethylsilyl)-trifluoroacetamide (DTSFA);
- 2-Hydrazinobenzothiazole (HBT).
6. Determination of Antioxidant Enzyme Activity
7. α-Tocopherol
8. Determination of the Most Informative Indicators of the Thiol–Disulfide System
9. Indicators of the Nitric Oxide System in the Brain
10. Assessment of Brain Energy Metabolism Indicators
11. Morphometry of Various Brain Structures
- Density of Neurons, Glial Cells, Apoptotic, and Destructively Altered Neurons: Measured as the number of cells per 1 mm2 of tissue section;
- Cellular Composition: Determined as the percentage of neurons, glial cells, apoptotic, and destructively altered neurons in the IV–V layers of the cortex and the CA1 region of the hippocampus;
- Area of Cell Bodies: Measured in µm2 for neurons, glial cells, apoptotic, and destructively altered neurons;
- RNA Concentration: In neurons, glial cells and apoptotic and destructively altered neurons are expressed in optical density units (ODU). This is calculated as the logarithm of the ratio of the optical density of the cell body to the optical density of the extracellular matrix;
- RNA Content: In neurons, glial cells and apoptotic and destructively altered neurons are expressed in ODU. This is calculated as the product of RNA concentration and cell area;
- Neuron Survival Index: Assessed as the ratio of the number of neurons in experimental animals to the number of neurons in intact control animals.
11.1. Markers with Informational Value in CNS Pathology
11.1.1. Gold Dot (NR2 Antibody Detection)
- Independent Serum Marker: The level of antibodies to NR2 serves as an independent serum marker for cerebral ischemic events;
- Neurotoxicity Marker: NR2 antibodies are indicative of neurotoxicity associated with ischemic damage;
- Monitoring Tool: Tracking NR2 antibody levels enables monitoring the efficacy of pharmacological interventions for ischemic brain injury.
11.1.2. Neuron-Specific Enolase (NSE) (Enzyme-Linked Immunosorbent Assay, Western Blot)
11.1.3. Myelin Basic Protein (MBP) (Enzyme-Linked Immunosorbent Assay, Western Blot)
11.1.4. S-100 Protein (Enzyme-Linked Immunosorbent Assay)
11.1.5. Galanin (Immunohistochemistry Enzyme-Linked Immunosorbent Assay, Western Blot)
11.1.6. Phosphorylated Neurofilament H (pNF-H) (Enzyme-Linked Immunosorbent Assay, Western Blot)
11.1.7. Glial Fibrillary Acidic Protein (GFAP) (Enzyme-Linked Immunosorbent Assay)
11.2. Markers of Neuroplasticity
11.2.1. Neurotrophin-3 (NT3) and Neurotrophin-4/5 (NT4/5) (Immunohistochemistry, Enzyme-Linked Immunosorbent Assay, Western Blot)
11.2.2. Expression of c-fos and c-jun Proteins in the Brain (Immunohistochemistry; Western Blot)
11.2.3. Expression/Concentration of Heat Shock Proteins in the Brain (Immunohistochemistry, Western Blot, Enzyme-Linked Immunosorbent Assay)
11.2.4. Hypoxia-Inducible Factor 1α (HIF-1α) (Immunohistochemistry, Immunoblotting, Enzyme-Linked Immunosorbent Assay)
11.2.5. Brain-Derived Neurotrophic Factor (BDNF) (Immunohistochemistry, Enzyme-Linked Immunosorbent Assay, Western Blot)
11.2.6. Ciliary Neurotrophic Factor (CNTF) (Immunohistochemistry, Enzyme-Linked Immunosorbent Assay, Western Blot)
11.2.7. Pigment Epithelium-Derived Factor (PEDF) (Immunohistochemistry, Western Blot)
11.3. Markers of Apoptosis
11.3.1. Annexin V (Immunohistochemistry, Western Blot)
11.3.2. Caspase-3 (Immunohistochemistry, Western Blot, Enzyme-Linked Immunosorbent Assay)
11.3.3. Cathepsins (Immunohistochemistry, Western Blot, Enzyme-Linked Immunosorbent Assay)
11.3.4. Procathepsin B (Immunohistochemistry, Western Blot, Enzyme-Linked Immunosorbent Assay)
11.3.5. DR5 (Death Receptor) (Immunohistochemistry, Western Blot)
11.3.6. Bcl-2 Family (Immunohistochemistry, Western Blot)
- Anti-apoptotic Subfamily: includes close homologs such as Bcl-2, Bcl-XL, and Bcl-w, which inhibit apoptosis;
- Pro-apoptotic Subfamilies: include proteins such as Bax and members of the BH3-only group, which promote apoptosis.
- Perform Preliminary Screening**: Evaluate a large number of molecules while preserving animal lives;
- Investigate Mechanisms**: Examine the impact of potential neuroprotectors on specific aspects of the ischemic brain damage pathogenesis, such as oxidative nitrosative stress, glutamate excitotoxicity, and thiol–disulfide balance shifts.
- Diffuse Decrease in Cerebral Blood Flow;
- Energy Deficit;
- Glutamate–Calcium Excitotoxicity;
- Oxidative Stress;
- Expression of Early Response Genes.
Author Contributions
Funding
Conflicts of Interest
References
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Group of Oxidative Stress Reaction Products | Chemical Compounds | Detection Methods |
---|---|---|
I. Unstable (Radical Nature) | Alkyl, alkoxyl, peroxyl, nitrite, peroxynitrite radicals | Spontaneous CL, Fe2+ or H2O2-induced CL, EPR |
II. Stable (Non-Radical Nature): | ||
(1) Primary | Hydroperoxides, conjugated dienes, endoperoxides, dialkyl peroxides, epoxides | Polarography, iodometry, UV, IR, NMR, HPLC |
(2) Secondary | Aldehydes (alkanal, alkenal), hydroxyalkenals, malondialdehyde, trienones, 8-isoprostanes, 8-hydroxy-2-deoxyguanosine, o-nitrotyrosine, o-chlorotyrosine, thymidine glycol | UV, HPLC, HPLC/Fluorescence, HPLC/UV, FS, TLC, GC, GC/MS |
(3) End-Products | Gaseous products (pentane, heptane, etc.), Schiff bases, nitrates, and nitrites | FS, GRP |
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Belenichev, I.; Bukhtiyarova, N.; Ryzhenko, V.; Makyeyeva, L.; Morozova, O.; Oksenych, V.; Kamyshnyi, O. Methodological Approaches to Experimental Evaluation of Neuroprotective Action of Potential Drugs. Int. J. Mol. Sci. 2024, 25, 10475. https://doi.org/10.3390/ijms251910475
Belenichev I, Bukhtiyarova N, Ryzhenko V, Makyeyeva L, Morozova O, Oksenych V, Kamyshnyi O. Methodological Approaches to Experimental Evaluation of Neuroprotective Action of Potential Drugs. International Journal of Molecular Sciences. 2024; 25(19):10475. https://doi.org/10.3390/ijms251910475
Chicago/Turabian StyleBelenichev, Igor, Nina Bukhtiyarova, Victor Ryzhenko, Lyudmyla Makyeyeva, Oksana Morozova, Valentyn Oksenych, and Oleksandr Kamyshnyi. 2024. "Methodological Approaches to Experimental Evaluation of Neuroprotective Action of Potential Drugs" International Journal of Molecular Sciences 25, no. 19: 10475. https://doi.org/10.3390/ijms251910475