The neurochemical profile of the hippocampus in isoflurane-treated and unanesthetized rat pups

Abstract

In vivo study of cerebral metabolism in neonatal animals by high-resolution magnetic resonance spectroscopy (MRS) is an important tool for deciphering the developmental origins of adult diseases. Up to date, all in vivo spectrum acquisition procedures have been performed in neonatal rodents under anesthesia. However, it is still unknown if the inhaled anesthetic isoflurane, which is commonly used in magnetic resonance imaging studies, could affect metabolite levels in the brain of neonatal rats. Moreover, the unanesthetized MRS preparation that uses neonatal rodent pups is still lacking. Here, a novel restraint protocol was developed for neonatal rats in accordance with the European Directive 2010/63/EU. This protocol shares the same gradation of severity as the protocol for non-invasive magnetic resonance imaging of animals with appropriate sedation or anesthesia. Such immobilization of neonatal rats without anesthesia can be implemented for MRS studies when an interaction between anesthetic and target drugs is expected. Short-term isoflurane treatment did not affect the levels of key metabolites in the hippocampi of anesthetized pups and, in contrast to juvenile and adult rodents, it is suitable for MRS studies in neonatal rats when the interaction between anesthetic and target drugs is not expected.

Keywords: hippocampus; isoflurane; magnetic resonance spectroscopy; neonatal rat; unanesthetized.

Figure 1

The immobilizing procedure for the neonatal rat pup. (see Materials and methods section for details).

Figure 2

Breathing frequency of pups and neurochemical profile of the hippocampi of untreated and isoflurane-treated neonatal rats on PD3. (a) Breathing frequency of 3-day-old rat pups during restraint procedure and under isoflurane anesthesia. *p<0.05 vs breathing frequency of untreated rats in min 6–30. **p<0.001 vs breathing frequency of untreated rats in min 6–30. #p<0.001 vs breathing frequency of isoflurane-treated pups at the same time point. Data are presented as the means±SEM. (b) Representative in vivo short-echo-time 1H NMR spectrum at 11.7T in the hippocampus of untreated neonatal rat (TE=3 ms, TM=20 ms, TR=4 s, 230 scans, 7.5-μL volume) and RARE image of the PD3 rat brain with the volume of interest centered in the hippocampus and selected for 1H NMR spectroscopy studies. (c) Measured ratios of compounds (metabolite/NAA) in the hippocampi of untreated and isoflurane-treated neonatal rats. Ala – alanine; Asp – aspartate; Cho – choline compounds; Cr+PCr – creatine + phosphocreatine; GABA – gamma-aminobuytric acid; Glu+Gln – glutamine + glutamate; Ins – myo-inositol; Lac – lactate; Tau – taurine. Data are presented as the means±SEM. None of the ratios differed significantly between experimental groups.