Exposure of neonatal rats to alcohol has differential effects on neuroinflammation and neuronal survival in the cerebellum and hippocampus methods
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Reverse transcriptase polymerase chain reaction
Samples were collected during peak BACs on P3 and P5 (within 30 min following the 4-h alcohol exposure) and during periods of alcohol withdrawal on P4 and P6 (Fig. ). Additionally, to address the duration of effects, samples were collected at P11-13. Eight pups (from a total of 8 litters) were used per treatment group. Animals were anesthetized with ketamine (250 mg/kg). Whole hippocampi and cerebellar vermises were collected. Tissue was homogenized by sonication in ice cold RNeasy Lysis Buffer and RNA was extracted using an RNeasy Mini Kit according to the manufacturer's instructions (SABiosciences/Qiagen, Valencia, CA). The RNA was stored at -20 °C until use. cDNA was synthesized from 1 µg of RNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, cat# 4368813, Foster City, CA) according to the manufacturer&...
Immunohistochemistry
On P4, P6, and P45 (Fig. ), 4 pups (from a total of 4 litters) per treatment group were anesthetized by injection of ketamine (250 mg/kg). Animals were transcardially perfused first with phosphate buffered saline (PBS), pH 7.4, containing procaine hydrochloride (1 g/L) and heparin (1 USP unit/L) for 4 min, followed by ice cold 4 % paraformaldehyde in PBS. Brains were removed by decapitation and placed in 4 % paraformaldehyde for 48 h at 4 °C, then 30 % sucrose for 24-48 h at 4 °C. Brains were embedded in Optimal Cutting Temperature compound (Fisher Healthcare, Houston, TX) and frozen before sectioning on a cryostat (Microm, model# HM 505E, Walldorf, Germany) at 16 µm. Sections for staining were incubated with PBS containing 1 % bovine serum albumin, 0.2 % Triton X-100, and 5 % of either do...
Gait analysis
Alterations in gait are associated with cerebellar impairment and particularly with Purkinje cell dysfunction [ - ]. Gait was assessed using the Catwalk XT system (Noldus, Wageningen, Netherlands) located at the Animal Behavioral Core of the Biomedical Research and Integrative Neuroimaging Center (BRaIN), UNM-HSC; two to three animals per litter were used from a total of 4 litters per treatment group. Rats aged P45-50 (Fig. ) were placed on an enclosed glass walkway illuminated from above by a red fluorescent light and along the walkway by green light-emitting diode lights. Disruption of the green light allowed for tracking of paw prints, while disruption of red light allowed for visualization of silhouettes. Rats were allowed to walk freely across the runway to a dark box, containing bedding from their home cage, located at the opposite end. A digital high-speed cam...
Measurement outputs
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Gait recovery and locomotor function
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
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- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
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Quantify the primary readouts for this experiment: Gait recovery and locomotor function.
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Report representative outputs alongside summary comparisons for Gait recovery and locomotor function.
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Evidence quotes (3)
Samples were collected during peak BACs on P3 and P5 (within 30 min following the 4-h alcohol exposure) and during periods of alcohol withdrawal on P4 and P6 (Fig. ). Additionally, to address the duration of effects, samples were collected at P11-13. Eight pups (from a total of 8 litters) were used per treatment group. Animals were anesthetized with ketamine (250 mg/kg). Whole hippocampi and cerebellar vermises were collected. Tissue was homogenized by sonication in ice cold RNeasy Lysis Buffer and RNA was extracted using an RNeasy Mini Kit according to the manufacturer's instructions (SABiosciences/Qiagen, Valencia, CA). The RNA was stored at -20 °C until use. cDNA was synthesized from 1 µg of RNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, cat# 4368813, Foster City, CA) according to the manufacturer's instructions. The following primers from SABiosciences/Qiagen were used: interleukin-1β (IL-1β, cat# PPR06480B), tumor necrosis factor α (TNFα, cat# PPR06411F), interleukin-10 (IL10, cat# PPR06479A), transforming growth factor β (TGFβ, cat# PPR06430B), and hypox...
On P4, P6, and P45 (Fig. ), 4 pups (from a total of 4 litters) per treatment group were anesthetized by injection of ketamine (250 mg/kg). Animals were transcardially perfused first with phosphate buffered saline (PBS), pH 7.4, containing procaine hydrochloride (1 g/L) and heparin (1 USP unit/L) for 4 min, followed by ice cold 4 % paraformaldehyde in PBS. Brains were removed by decapitation and placed in 4 % paraformaldehyde for 48 h at 4 °C, then 30 % sucrose for 24-48 h at 4 °C. Brains were embedded in Optimal Cutting Temperature compound (Fisher Healthcare, Houston, TX) and frozen before sectioning on a cryostat (Microm, model# HM 505E, Walldorf, Germany) at 16 µm. Sections for staining were incubated with PBS containing 1 % bovine serum albumin, 0.2 % Triton X-100, and 5 % of either donkey or goat serum to match the host species of the secondary antibodies described below. Primary antibodies were applied overnight at 4 °C. Astrocytes were stained with rabbit anti-glial fibrillary acidic protein (GFAP, 1:500, Dako, cat# 019-19741, Carpinteria, CA), microglia with rabbit...
Alterations in gait are associated with cerebellar impairment and particularly with Purkinje cell dysfunction [ - ]. Gait was assessed using the Catwalk XT system (Noldus, Wageningen, Netherlands) located at the Animal Behavioral Core of the Biomedical Research and Integrative Neuroimaging Center (BRaIN), UNM-HSC; two to three animals per litter were used from a total of 4 litters per treatment group. Rats aged P45-50 (Fig. ) were placed on an enclosed glass walkway illuminated from above by a red fluorescent light and along the walkway by green light-emitting diode lights. Disruption of the green light allowed for tracking of paw prints, while disruption of red light allowed for visualization of silhouettes. Rats were allowed to walk freely across the runway to a dark box, containing bedding from their home cage, located at the opposite end. A digital high-speed camera recorded each trial until three compliant trials were captured in which the animal did not stop or turn around and crossed the walkway within 0.5-10 s with a maximum body speed variation of less than 60 %. PAE significantly increased the total number of trials required to achieve...
Machine-readable layer
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