Muhammad Rafiq, PhD
Author affiliation:
Department of Psychology, Lahore School of Professional Studies, The University of Lahore, Lahore–Pakistan.
Correspondence: Dr. Muhammad Rafiq, PhD, Department of Psychology, Lahore School of Professional Studies, The University of Lahore, 1–Km Defence Road،, near Bhuptian Chowk, Lahore Lahore–Pakistan. E–mail: rafiqdar@hotmail.com
Abstract
Background & objective: Previous studies showed daily variations of brain–derived neurotropic factor (BDNF) but there is no evidence of any circadian rhythm of BDNF proteins and still there is no data regarding effect of propofol anesthesia on the circadian rhythmicity of BDNF protein in the brain. This study examined the effect of propofol anesthesia on circadian rhythm of BDNF protein in hippocampus and cortex.
Methodology: Male rats were treated with propofol anaesthesia (120 mg/kg) with their intralipid control. Subjects were sacrificed at four different zeitgeber times (ZT5, ZT11, ZT17 and ZT23). Here, ZT0 and ZT12 defining lights on and off respectively. The brains were removed and brain homogenates were prepared from hippocampus and cortex tissues. The amount of BDNF protein was assessed using ELISA (Millipore) on the brain supernatants.
Results: The main results showed that this circadian variation of BDNF protein in both the hippocampal and cortical structures is disturbed by the propofol anesthesia. Post–hoc analyses showed that animals treated with intralipid as control showed an effect on daily variation of BDNF in both hippocampal and cortical regions (all have P<0.01).
Conclusion: Our results provided the first evidence that BDNF protein expression follows a physiological circadian rhythm in hippocampus and cortex with a maximum activity evidenced at ZT5 (5h after light on) and for the first time, we evidenced that this circadian rhythm of BDNF protein is dysregulated by the propofol anaesthesia.
Key words: Circadian rhythms; Propofol; Cortex; Hippocampus; ELISA; BDNF
Abbreviations: BDNF – Brain–derived neurotrophic factor; RT–PCR – Reverse transcription polymerase chain reaction
Citation: Rafiq M. Effect of propofol on circadian variation of brain–derived neurotrophic factor. Anaesth. pain intensive care 2021;25(3):345–348. DOI: 10.35975/apic.v25i3.1528
Received: April 28, 2019; Reviewed: February 26, March 31, 2021; Accepted: March 31, 2021
Introduction
Brain–derived neurotrophic factor (BDNF) is a member of the neurotrophin family and is expressed in all areas of the mammalian brain especially hippocampus and cortex. It is observed that hippocampus and cortex contain the highest level of BDNF mRNA as compared to nerve growth factor1. BDNF is suggested to play important role in synaptic plasticity and memory performance.2 Previous studies have suggested that BDNF central levels are influenced by light and dark. Diurnal changes of BDNF mRNA contents have been demonstrated in the rat central nervous system. Reverse transcription polymerase chain reaction (RT–PCR) shows BDNF mRNA variations at four different times in adult rats during a 24–h cycle with the strongest variation in the hippocampus.3 Plasma BDNF protein has been shown to vary at different times of the day in human males with a peak value in the morning and is decreased during the day.4 In situ hybridization shows BDNF mRNA variation during 24 hours of the day at four time–points (noon, 6 pm, midnight and 6 am) in hippocampus of rats.5 Hippocampal subfields (DG, CA1 and CA3) show also BDNF mRNA variation during 12/12 hour light/dark cycle.6
Anesthetics are known to cause sedation and disturb sleep/wake cycle. Propofol anesthesia induces amnesia not only at low dose under its administration in both animal models and in human volunteers but also at recovery from anesthesia.7 Circadian rest–activity rhythms are disturbed by the general anesthetics8 and Propofol is involved in dysregulation of circadian rhythms of melatonin under normal light/dark conditions.9 The above studies suggest that propofol may dysregulate BDNF protein circadian rhythms in the hippocampus and cortex of the rats. This is the main rationale of the study. As BDNF has been implicated in different biological processes including learning, memory etc. So, any effect of propofol anesthesia on daily variation of BDNF might alter the BDNF bases processes including learning and memory. In addition, it may also reflect the daytime where propofol has minimum effect.
So goal of this study was to observe whether there is dysregulation of BDNF protein circadian by the short duration anesthesia propofol or not. For this purpose, animals were either injected with propofol anesthesia (120 mg/kg) at four different zeitgeber times (ZT5, ZT11, ZT17 and ZT23) or with intralipid (Intralipid® (Fresenius, France) as control.
Methodology
All the experiments were conducted following institutional guidelines complying with national and international guidelines at Institute of Molecular and Integrative Neurosciences, University of Strasbourg, France.
Young male Sprague Dawley rats were housed in temperature (20 ± 1C) and humidity (50 ± 10%) on a twelve–hour light/dark cycle (lights on 7:00 am), with ad libitum access to food and water. Animals were divided into four groups (each group of 16) based of zeitgeber time (ZT5, ZT11, ZT13 and ZT23). At each zeitgeber time, 8 subjects were injected intraperitoneally with propofol (Fresenius, France) at a rate of 120 mg/kg and 8 with intralipid as a control. Animals at each zeitgeber time were sacrificed after euthanasia under CO2 at the concentration of 0.5 bars for maximum one min. Brains were quickly removed on ice; and cortex and hippocampal structures were taken. The tissues were homogenized in extraction buffer and frozen at –20° C till BDNF protein quantification.
Prior to BDNF analysis, protein contents were determined using a protein assay kit (Bio–Rad). BDNF protein was quantified using a BDNF ELISA kit validated for rat and human BDNF detection (CYT306, Millipore) according to the manufacturer’s recommendations.10, 11
Briefly, 100 µl of samples diluted (1:2) with the sample diluent provided in the kit were loaded in the 96 wells ELISA plate and incubated 12h at 4C°. The plates were washed (300 µl) four times with wash buffer provided in the kit. Then 100 µl of anti–BDNF antibody (1/1000) were added in each well and incubated for 3hours at room temperature. Wells were washed with four times with wash buffer and 100 µl of streptavidin HRP (1:1000) were added and incubated for one hour at room temperature. Wells were washed four times with wash buffer and coloration was done for 15 min with 100 µl of TBM. The reaction was blocked by adding 100 µl of stop solution and ELISA plates were read at 450nm. For all tests, BDNF standards were diluted in the sample diluting buffer and extraction buffer (1:1, as for the sample). Standard and samples were loaded in duplicates. The CV values were between 0 to 8%. All the samples with a higher CV value were retested to obtain a CV below or equal to 8%. The experimental range detection of 7.8 pg/ml to 500 g/ml obtained for the unique kit batch used was following ELISA kit’s datasheet.
Results
We observed a circadian variation of BDNF protein in hippocampus and cortical structures of rats’ brain. This circadian rhythmicity of BDNF protein at four time–points (ZT5, ZT11, ZT17 and ZT23) is observed maximum at ZT5 (rest period) and nadir at ZT17 (activity period). BDNF protein is significantly varied at these two transitory times (Figure 1 and Figure 2). This circadian variation of BDNF protein observed with intralipid in both the hippocampal and cortical structures is significantly disturbed by the short duration propofol anesthesia (Figure 2 and Figure 2a).
Post–hoc analysis showed that animals submitted to intralipid as control show a significant variation of BDNF protein at zeitgeber times between light and dark periods in both hippocampal and cortical regions (P < 0.01). This significant variation of BDNF protein is totally disturbed by the propofol anesthesia.
Discussion
In the present study, we observed daily variation of BDNF protein in the rat hippocampus and prefrontal cortex at four different zeitgeber times (ZT5, ZT11, and ZT17 and ZT23). This variation was observed when animals were submitted to intralipid as control and it has no effect on the normal physiology of daily variation of BDNF protein. This daily variation of BDNF protein was altered when animals were submitted to short–duration propofol anesthesia. The BDNF protein in the cortex and hippocampal supernatants was quantified with ELISA technique. ELISA is a quick and convenient method for the determination of antigens in tissue homogenates and this has been shown to achieve sensitivities and specificities.12
Data in the literature show that the BNDF central levels are altered by the light and dark periods. RT–PCR determined that the BDNF mRNA is varied during the 24 hour period measured at four time-points in the hippocampus and cortical structures of the rat’s brain3. In this study, mRNA is at a peak at ZT13 (one hour at the start of the activity period) and minimum at ZT5 (Five hours at start of rest period). Rat’s hippocampal subfields (CA1, CA3 and DG) have shown BDNF mRNA variation at four time–points and BDNF is observed maximum during the dark period and nadir during the light period.5 In situ hybridization, BDNF mRNA expression significantly varies in different hippocampal subfields (DG, CA1 and CA3) at different time–points during 12/12 h light/dark cycle.6 This
study has also shown that during the dark cycle (activity period), BDNF mRNA is increased and during the light cycle (rest period), BDNF mRNA is decreased.
The above studies show incompatibilities with our results, as the above studies showed globally that BDNF mRNA is increased during the activity period and decreased during the rest period. In contrary to this, our results showed that during the activity period BDNF protein is decreased and during the rest period, BDNF protein is increased. This may be due to the differences in mRNA and protein turnover rates, as the central dogma of molecular biology states that DNA transcribes mRNA and mRNA is translated into proteins, suggesting that there is a direct relationship between mRNA and protein levels but it is also observed that biologically, variation between mRNA and protein expression level can be caused by posttranscriptional regulation as well as differences in mRNA and protein turnover rates.13 It may be possible that variation between mRNA and protein levels may be caused by circadian time.
Few studies describe the effect of anesthetics on the circadian rhythms of BDNF protein. However, some studies evidenced that anesthetics have effects on circadian rhythm. It is noted that there is circadian rhythmicity of duration of loss of righting reflex when animals were submitted to propofol (100 mg/kg) at six different time points of the day14. BDNF is directly linked to the activity and any stimuli that are responsible to disturb the activity may also disturb the BDNF protein. Dispersyn et al. (2009) evidenced that the circadian rest–activity rhythms are disturbed by the anesthetics and it is also observed the dysregulation of circadian rhythms of melatonin by the short duration anesthesia under normal light/ dark conditions.9 These studies show compatibilities with our results that show a dysregulation of BDNF protein by the short duration anesthesia propofol. Despite the interesting results, there are few limitations as well i.e. our data cannot be generalized on long–duration anesthetics and/or other neurotropic factors. In this study, only young brains were used, so we cannot conclude our results on the old brains.
Perspectives
Propofol anesthesia induces amnesia not only at a low dose under its administration in both animal models and in human volunteers but also at recovery from anesthesia.7 BDNF has been shown to be involved in synaptic plasticity and memory performance. It is shown that PERK1/2 is also involved in synaptic plasticity and memory performance and it has been studied that PERK1/2 protein shows a circadian rhythm as well. 15 PERK1/2 protein peak is observed at around ZT5 (parallel to peak of BDNF protein at ZT5). This circadian rhythmicity of PERK1/2 is parallel to the circadian memory performance, 16 as the maximum memory performance is observed at around ZT5. There are few studies available on the effect of propofol on the circadian rhythmicity of memory, so yet to be examined. For this, it will be necessary to assess the memory performance after submitting propofol at different time points during the day.
Conflict of interest
No funding was involved in this study.
Author’s contribution
MR: Research, Data analysis, manuscript preparation
References
Author affiliation:
Department of Psychology, Lahore School of Professional Studies, The University of Lahore, Lahore–Pakistan.
Correspondence: Dr. Muhammad Rafiq, PhD, Department of Psychology, Lahore School of Professional Studies, The University of Lahore, 1–Km Defence Road،, near Bhuptian Chowk, Lahore Lahore–Pakistan. E–mail: rafiqdar@hotmail.com
Abstract
Background & objective: Previous studies showed daily variations of brain–derived neurotropic factor (BDNF) but there is no evidence of any circadian rhythm of BDNF proteins and still there is no data regarding effect of propofol anesthesia on the circadian rhythmicity of BDNF protein in the brain. This study examined the effect of propofol anesthesia on circadian rhythm of BDNF protein in hippocampus and cortex.
Methodology: Male rats were treated with propofol anaesthesia (120 mg/kg) with their intralipid control. Subjects were sacrificed at four different zeitgeber times (ZT5, ZT11, ZT17 and ZT23). Here, ZT0 and ZT12 defining lights on and off respectively. The brains were removed and brain homogenates were prepared from hippocampus and cortex tissues. The amount of BDNF protein was assessed using ELISA (Millipore) on the brain supernatants.
Results: The main results showed that this circadian variation of BDNF protein in both the hippocampal and cortical structures is disturbed by the propofol anesthesia. Post–hoc analyses showed that animals treated with intralipid as control showed an effect on daily variation of BDNF in both hippocampal and cortical regions (all have P<0.01).
Conclusion: Our results provided the first evidence that BDNF protein expression follows a physiological circadian rhythm in hippocampus and cortex with a maximum activity evidenced at ZT5 (5h after light on) and for the first time, we evidenced that this circadian rhythm of BDNF protein is dysregulated by the propofol anaesthesia.
Key words: Circadian rhythms; Propofol; Cortex; Hippocampus; ELISA; BDNF
Abbreviations: BDNF – Brain–derived neurotrophic factor; RT–PCR – Reverse transcription polymerase chain reaction
Citation: Rafiq M. Effect of propofol on circadian variation of brain–derived neurotrophic factor. Anaesth. pain intensive care 2021;25(3):345–348. DOI: 10.35975/apic.v25i3.1528
Received: April 28, 2019; Reviewed: February 26, March 31, 2021; Accepted: March 31, 2021
Introduction
Brain–derived neurotrophic factor (BDNF) is a member of the neurotrophin family and is expressed in all areas of the mammalian brain especially hippocampus and cortex. It is observed that hippocampus and cortex contain the highest level of BDNF mRNA as compared to nerve growth factor1. BDNF is suggested to play important role in synaptic plasticity and memory performance.2 Previous studies have suggested that BDNF central levels are influenced by light and dark. Diurnal changes of BDNF mRNA contents have been demonstrated in the rat central nervous system. Reverse transcription polymerase chain reaction (RT–PCR) shows BDNF mRNA variations at four different times in adult rats during a 24–h cycle with the strongest variation in the hippocampus.3 Plasma BDNF protein has been shown to vary at different times of the day in human males with a peak value in the morning and is decreased during the day.4 In situ hybridization shows BDNF mRNA variation during 24 hours of the day at four time–points (noon, 6 pm, midnight and 6 am) in hippocampus of rats.5 Hippocampal subfields (DG, CA1 and CA3) show also BDNF mRNA variation during 12/12 hour light/dark cycle.6
Anesthetics are known to cause sedation and disturb sleep/wake cycle. Propofol anesthesia induces amnesia not only at low dose under its administration in both animal models and in human volunteers but also at recovery from anesthesia.7 Circadian rest–activity rhythms are disturbed by the general anesthetics8 and Propofol is involved in dysregulation of circadian rhythms of melatonin under normal light/dark conditions.9 The above studies suggest that propofol may dysregulate BDNF protein circadian rhythms in the hippocampus and cortex of the rats. This is the main rationale of the study. As BDNF has been implicated in different biological processes including learning, memory etc. So, any effect of propofol anesthesia on daily variation of BDNF might alter the BDNF bases processes including learning and memory. In addition, it may also reflect the daytime where propofol has minimum effect.
So goal of this study was to observe whether there is dysregulation of BDNF protein circadian by the short duration anesthesia propofol or not. For this purpose, animals were either injected with propofol anesthesia (120 mg/kg) at four different zeitgeber times (ZT5, ZT11, ZT17 and ZT23) or with intralipid (Intralipid® (Fresenius, France) as control.
Methodology
All the experiments were conducted following institutional guidelines complying with national and international guidelines at Institute of Molecular and Integrative Neurosciences, University of Strasbourg, France.
Young male Sprague Dawley rats were housed in temperature (20 ± 1C) and humidity (50 ± 10%) on a twelve–hour light/dark cycle (lights on 7:00 am), with ad libitum access to food and water. Animals were divided into four groups (each group of 16) based of zeitgeber time (ZT5, ZT11, ZT13 and ZT23). At each zeitgeber time, 8 subjects were injected intraperitoneally with propofol (Fresenius, France) at a rate of 120 mg/kg and 8 with intralipid as a control. Animals at each zeitgeber time were sacrificed after euthanasia under CO2 at the concentration of 0.5 bars for maximum one min. Brains were quickly removed on ice; and cortex and hippocampal structures were taken. The tissues were homogenized in extraction buffer and frozen at –20° C till BDNF protein quantification.
Prior to BDNF analysis, protein contents were determined using a protein assay kit (Bio–Rad). BDNF protein was quantified using a BDNF ELISA kit validated for rat and human BDNF detection (CYT306, Millipore) according to the manufacturer’s recommendations.10, 11
Briefly, 100 µl of samples diluted (1:2) with the sample diluent provided in the kit were loaded in the 96 wells ELISA plate and incubated 12h at 4C°. The plates were washed (300 µl) four times with wash buffer provided in the kit. Then 100 µl of anti–BDNF antibody (1/1000) were added in each well and incubated for 3hours at room temperature. Wells were washed with four times with wash buffer and 100 µl of streptavidin HRP (1:1000) were added and incubated for one hour at room temperature. Wells were washed four times with wash buffer and coloration was done for 15 min with 100 µl of TBM. The reaction was blocked by adding 100 µl of stop solution and ELISA plates were read at 450nm. For all tests, BDNF standards were diluted in the sample diluting buffer and extraction buffer (1:1, as for the sample). Standard and samples were loaded in duplicates. The CV values were between 0 to 8%. All the samples with a higher CV value were retested to obtain a CV below or equal to 8%. The experimental range detection of 7.8 pg/ml to 500 g/ml obtained for the unique kit batch used was following ELISA kit’s datasheet.
Results
We observed a circadian variation of BDNF protein in hippocampus and cortical structures of rats’ brain. This circadian rhythmicity of BDNF protein at four time–points (ZT5, ZT11, ZT17 and ZT23) is observed maximum at ZT5 (rest period) and nadir at ZT17 (activity period). BDNF protein is significantly varied at these two transitory times (Figure 1 and Figure 2). This circadian variation of BDNF protein observed with intralipid in both the hippocampal and cortical structures is significantly disturbed by the short duration propofol anesthesia (Figure 2 and Figure 2a).
Post–hoc analysis showed that animals submitted to intralipid as control show a significant variation of BDNF protein at zeitgeber times between light and dark periods in both hippocampal and cortical regions (P < 0.01). This significant variation of BDNF protein is totally disturbed by the propofol anesthesia.
Discussion
In the present study, we observed daily variation of BDNF protein in the rat hippocampus and prefrontal cortex at four different zeitgeber times (ZT5, ZT11, and ZT17 and ZT23). This variation was observed when animals were submitted to intralipid as control and it has no effect on the normal physiology of daily variation of BDNF protein. This daily variation of BDNF protein was altered when animals were submitted to short–duration propofol anesthesia. The BDNF protein in the cortex and hippocampal supernatants was quantified with ELISA technique. ELISA is a quick and convenient method for the determination of antigens in tissue homogenates and this has been shown to achieve sensitivities and specificities.12
Data in the literature show that the BNDF central levels are altered by the light and dark periods. RT–PCR determined that the BDNF mRNA is varied during the 24 hour period measured at four time-points in the hippocampus and cortical structures of the rat’s brain3. In this study, mRNA is at a peak at ZT13 (one hour at the start of the activity period) and minimum at ZT5 (Five hours at start of rest period). Rat’s hippocampal subfields (CA1, CA3 and DG) have shown BDNF mRNA variation at four time–points and BDNF is observed maximum during the dark period and nadir during the light period.5 In situ hybridization, BDNF mRNA expression significantly varies in different hippocampal subfields (DG, CA1 and CA3) at different time–points during 12/12 h light/dark cycle.6 This
| Figure 2a: Dysregulated variation of BDNF protein in rat’s cortex during 24 h of the day, after injection of propofol anesthesia. |
The above studies show incompatibilities with our results, as the above studies showed globally that BDNF mRNA is increased during the activity period and decreased during the rest period. In contrary to this, our results showed that during the activity period BDNF protein is decreased and during the rest period, BDNF protein is increased. This may be due to the differences in mRNA and protein turnover rates, as the central dogma of molecular biology states that DNA transcribes mRNA and mRNA is translated into proteins, suggesting that there is a direct relationship between mRNA and protein levels but it is also observed that biologically, variation between mRNA and protein expression level can be caused by posttranscriptional regulation as well as differences in mRNA and protein turnover rates.13 It may be possible that variation between mRNA and protein levels may be caused by circadian time.
Few studies describe the effect of anesthetics on the circadian rhythms of BDNF protein. However, some studies evidenced that anesthetics have effects on circadian rhythm. It is noted that there is circadian rhythmicity of duration of loss of righting reflex when animals were submitted to propofol (100 mg/kg) at six different time points of the day14. BDNF is directly linked to the activity and any stimuli that are responsible to disturb the activity may also disturb the BDNF protein. Dispersyn et al. (2009) evidenced that the circadian rest–activity rhythms are disturbed by the anesthetics and it is also observed the dysregulation of circadian rhythms of melatonin by the short duration anesthesia under normal light/ dark conditions.9 These studies show compatibilities with our results that show a dysregulation of BDNF protein by the short duration anesthesia propofol. Despite the interesting results, there are few limitations as well i.e. our data cannot be generalized on long–duration anesthetics and/or other neurotropic factors. In this study, only young brains were used, so we cannot conclude our results on the old brains.
Perspectives
Propofol anesthesia induces amnesia not only at a low dose under its administration in both animal models and in human volunteers but also at recovery from anesthesia.7 BDNF has been shown to be involved in synaptic plasticity and memory performance. It is shown that PERK1/2 is also involved in synaptic plasticity and memory performance and it has been studied that PERK1/2 protein shows a circadian rhythm as well. 15 PERK1/2 protein peak is observed at around ZT5 (parallel to peak of BDNF protein at ZT5). This circadian rhythmicity of PERK1/2 is parallel to the circadian memory performance, 16 as the maximum memory performance is observed at around ZT5. There are few studies available on the effect of propofol on the circadian rhythmicity of memory, so yet to be examined. For this, it will be necessary to assess the memory performance after submitting propofol at different time points during the day.
Conflict of interest
No funding was involved in this study.
Author’s contribution
MR: Research, Data analysis, manuscript preparation
References
- Hofer M, Pagliusi SR, Hohn A, Leibrock J, Barde YA. Regional distribution of brain–derived neurotrophic factor mRNA in the adult mouse brain. EMBO J. 1990 Aug;9(8):2459–64. [PubMed] PMCID: PMC552273
- Figurov A, Pozzo–Miller LD, Olafsson P, Wang T, Lu B. Regulation of synaptic responses to high–frequency stimulation and LTP by neurotrophins in the hippocampus. Nature. 1996 Jun 20;381(6584):706–9. [PubMed] DOI: 1038/381706a0
- Bova R, Micheli MR, Qualadrucci P, Zucconi GG. BDNF and trkB mRNAs oscillate in rat brain during the light–dark cycle. Brain Res Mol Brain Res. 1998 Jun 15;57(2):321–4. [PubMed] DOI: 1016/s0169–328x(98)00092–8
- Begliuomini S, Lenzi E, Ninni F, Casarosa E, Merlini S, Pluchino N, Valentino V, Luisi S, Luisi M, Genazzani AR. Plasma brain–derived neurotrophic factor daily variations in men: correlation with cortisol circadian rhythm. J Endocrinol. 2008 May;197(2):429–35. [PubMed] DOI: 1677/JOE–07–0376
- Berchtold NC, Oliff HS, Isackson P, Cotman CW. Hippocampal BDNF mRNA shows a diurnal regulation, primarily in the exon III transcript. Brain Res Mol Brain Res. 1999 Jul 23;71(1):11–22. [PubMed]DOI: 1016/s0169–328x(99)00137–0
- Schaaf MJ, Duurland R, de Kloet ER, Vreugdenhil E. Circadian variation in BDNF mRNA expression in the rat hippocampus. Brain Res Mol Brain Res. 2000 Feb 22;75(2):342–4. [PubMed] DOI: 1016/s0169–328x(99)00314–9
- Pain L, Angst MJ, LeGourrier L, Oberling P. Effect of a nonsedative dose of propofol on memory for aversively loaded information in rats. Anesthesiology. 2002 Aug;97(2):447–53. [PubMed] DOI: 1097/00000542–200208000–00023 Dispersyn G, Touitou Y, Coste O, Jouffroy L, Lleu JC, Challet E, Pain L. Desynchronization of daily rest–activity rhythm in the days following light propofol anesthesia for colonoscopy. Clin Pharmacol Ther. 2009 Jan;85(1):51–5.. [PubMed] DOI: 10.1038/clpt.2008.179
- Dispersyn G, Pain L, Touitou Y. Propofol anesthesia significantly alters plasma blood levels of melatonin in rats. Anesthesiology. 2010 Feb;112(2):333–7. [PubMed]DOI: 1097/ALN.0b013e3181c920e2
- Kozisek ME, Middlemas D, Bylund DB. The differential regulation of BDNF and TrkB levels in juvenile rats after four days of escitalopram and desipramine treatment. Neuropharmacology. 2008 Feb;54(2):251–7. [PubMed] DOI: 1016/j.neuropharm.2007.08.001
- Wei X, Du Z, Zhao L, Feng D, Wei G, He Y, Tan J, Lee WH, Hampel H, Dodel R, Johnstone BH, March KL, Farlow MR, Du Y. IFATS collection: The conditioned media of adipose stromal cells protect against hypoxia–ischemia–induced brain damage in neonatal rats. Stem Cells. 2009 Feb;27(2):478–88. [PubMed] DOI: 1634/stemcells.2008–0333
- Spiehler VR, Collison IB, Sedgwick PR, Perez SL, Le SD, Farnin DA. Validation of an automated microplate enzyme immunoassay for screening of postmortem blood for drugs of abuse. J Anal Toxicol. 1998 Nov–Dec;22(7):573–9. [PubMed] DOI: 1093/jat/22.7.573
- Cox B, Kislinger T, Emili A. Integrating gene and protein expression data: pattern analysis and profile mining. Methods. 2005 Mar;35(3):303–14. doi: 10.1016/j.ymeth.2004.08.021. [PubMed] DOI: 1016/j.ymeth.2004.08.021
- Challet E, Gourmelen S, Pevet P, Oberling P, Pain L. Reciprocal relationships between general (Propofol) anesthesia and circadian time in rats. Neuropsychopharmacology. 2007 Mar;32(3):728–35. [PubMed] DOI: 1038/sj.npp.1301081
- Eckel–Mahan KL, Phan T, Han S, Wang H, Chan GC, Scheiner ZS, Storm DR. Circadian oscillation of hippocampal MAPK activity and cAmp: implications for memory persistence. Nat Neurosci. 2008 Sep;11(9):1074–82. [PubMed] DOI: 1038/nn.2174
- Roth TL, Sweatt JD. Rhythms of memory. Nat Neurosci. 2008 Sep;11(9):993–4. [PubMed] DOI: 10.1038/nn0908–993