abstract results results results conclusions funding design
Ketamine Alters Hippocampal Cell Proliferation after Traumatic Brain Injury and Improves Learning in Mice Austin J Peters1, Laura E Villasana1, Eric Schnell1,2 1Oregon Health & Science University, Department of Anesthesiology & Perioperative Medicine, Portland, OR 2VA Portland Health Care System, Anesthesiology Service, Portland, OR
ABSTRACT A
RESULTS
RESULTS
Ketamine Increases Hippocampal Cell Proliferation After CCI
Ketamine Inhibits Astrogliogenesis After CCI
Ketamine Ameliorates the CCI-Induced Impairment in Water Maze Reversal Learning
Vehicle
Ketamine
B
3 Days Post-CCI Sham + Vehicle
CCI + Vehicle
2 Weeks Post-CCI Sham + Vehicle
CCI + Vehicle
Sham
Sham + Ketamine
Sham + Ketamine
DAPI NeuN
C
DAPI BrdU
6 Weeks Post-CCI Sham + Vehicle
CCI + Vehicle
D
BrdU+ Cells
C DAPI GFAP Sham + Ketamine
CCI + Ketamine
(A) Representative images of Sham (non-injured) and CCI-injured coronal sections at 2 weeks post-injury. (B) Ketamine administration after CCI induced a robust increase in newly born (BrdU+, red) cells in the granule cell layer 3 days after injury. (C) Images of BrdU-stained dentate gyrus taken 6 weeks after CCI, demonstrating increased BrdU staining. (D) Quantification of BrdU+ labeled cells over time at the various time points evaluated in this study.
Ketamine Treatment Facilitates Rapid Transient Microgliogenesis After A B 3 Days Post-CCI CCI Sham + Vehicle
2 Weeks Post-CCI Sham + Vehicle
CCI + Vehicle
Sham + Ketamine
CCI + Ketamine
C
DAPI BrdU Iba1
Ketamine Does Not Modulate Other Indicators of Injury Severity Injury Sizes After CCI
Sham + Ketamine
CCI + Ketamine
CCI + Ketamine
B
6 WPI
IHC
C
C-fos Expression 3 Days Post-CCI Sham + Vehicle
CCI + Vehicle
Sham + Ketamine
CCI + Ketamine
D
E
DAPI cfos
(A) Representational images of the granule cell layer of the 4 treatment groups at 6 weeks after injury. Dcx+/BrdU+ co-labeling (red arrows) represents either continued mitotic activity of cells born after injury or persistent expression of the immature neuronal marker Dcx. (B) CCI decreased Dcx labeling across treatment groups with no change in ketamine vs. vehicle exposure. (C) Dcx+/BrdU+ co-labeled cells were not significantly increased between individual groups, but there was an overall significant increase Dcx+/BrdU+ co-labeling in all CCI groups versus Sham groups.
•Ketamine after TBI induces rapid early cellular proliferation (largely composed of microgliogenesis) and promotes survival of cells. •Ketamine transiently reduces astrogliogenesis and neurogenesis. •Ketamine rescues MWM reversal task performance after TBI. •TBI induces long term suppression of neurogenesis. •Improved MWM reversal may be related to the observed cellular responses, subtle anti-inflammatory changes not seen in this study, or off-target receptor activity by ketamine. Additional studies focusing on cellular mechanisms of ketamine and hippocampal stem cell responses to TBI are needed.
2 WPI
CCI + Vehicle
DAPI NeuN BrdU
Week 6
Week 4
Forced Swim Test Results
C
CCI + Ketamine
(A) Representative images of the GCL (DAPI, blue), microglia (Iba1+, white) and newly generated BrdU-labeled cells (green) at 3 days after intervention. Newly generated microglia (Iba1+/BrdU+) were greatly increased only in the Ketamine + CCI group at this time point. (B) Quantification of microgliogenesis 3 days after injury demonstrated a significant increase only in the Ketamine + CCI group. (C) At 6 weeks post-intervention, post-CCI-born microglia were significantly increased in both CCI groups versus their respective Sham groups.
CCI Delays the Maturation of Adult Born Neurons and Suppresses Hippocampal Neurogenesis Independent of Ketamine Behavioral Testing
IHC Week 2
Pump removal Day 7
BrdU BrdU vs IHC Day 2 Day 3
D
CONCLUSIONS
A
Intervention
CCI + Vehicle
(A) All groups successfully demonstrated preference for the target location in the Morris Water Maze (MWM), indicating grossly intact hippocampal function. (B) In the reversal task of the MWM, the CCI + Vehicle group failed to adapt to the new location, whereas the CCI + Ketamine group was able to achieve the task. (C) Characteristic tracings of each group from the reversal probe trials. (D) In the Forced Swim Test, ketamine administration did not affect the performance of CCI-induced mice.
B
(A) Representative images from each treatment group at 2 weeks post-injury. Immature neurons are stained with Dcx (white), along with BrdU-labeled cells (red) and DAPI (blue). Arrows indicate BrdU+/Dcx+ co-labeled cells. (B) Quantification of Dcx staining at 2 WPI demonstrated that ketamine administration suppressed neurogenesis after CCI. (C) Ketamine administration suppressed the number of neurons born 2-3 days after injury, as identified using the density of BrdU+/Dcx+ co-labaled cells.
Day 0
Sham + Vehicle
CCI + Vehicle
Post-Injury Ketamine Inhibits Neurogenesis 2 Weeks After CCI A
MWM Reversal Pathway Tracings
(A) Representative images of the four treatment groups at 2 weeks after intervention. The CCI + Vehicle group demonstrated a robust astrocytic response (GFAP+, red) not seen in the CCI + Ketamine group. (B) Quantification of astrogliogenesis 2 weeks post-injury demonstrated a significant increase in new astroglia (GFAP+/Dcx+ cells) in the Vehicle + CCI group that was blocked by ketamine.
DAPI BrdU Dcx
Mice were randomly assigned to receive CCI or Sham (no-injury), and subsequently implanted with an osmotic drug pump delivering ketamine or vehicle (saline). Dividing cells were labeled with BrdU at 2 and/or 3 days after the intervention. Immunohistochemical assays were employed to evaluate cellular responses in the granule cell layer of the hippocampus at 3 days, 2 weeks and 6 weeks after the intervention. Behavioral testing was conducted 4 weeks after the intervention.
Morris Water Maze Reversal Results
CCI + Ketamine
Sham + Ketamine
DESIGN
B
CCI + Ketamine
DAPI BrdU
Conclusions Ketamine alters cell proliferation after traumatic brain injury and suppresses constitutive and post-injury neurogenesis. Surprisingly, it improved behavioral outcomes specific to neurogenesis-related tasks, suggesting that the benefit of this treatment may have been due to ketamine-induced qualitative, and not quantitative effects on neurogenesis or due to changes in the glial modulation of neuronal circuit function.
Morris Water Maze (MWM) Results
B
Methods TBI was modeled in mice using controlled cortical impact (CCI) followed by a one-week continuous infusion of either vehicle or ketamine by osmotic pump. We utilized immunohistochemical and behavioral assays to evaluate cellular responses and recovery at different time points after injury. Results Ketamine treatment significantly increased hippocampal cell proliferation at early and late time points after CCI. Ketamine treatment after CCI also increased early microglial proliferation and reduced astrogliogenesis. Independent of injury, ketamine temporarily reduced neurogenesis. Ketamine administration rescued hippocampus dependent context-separation deficits of injured mice, as assayed by the Morris Water Maze reversal test.
A
A
2 WPI
Background Traumatic brain injury (TBI) is associated with long-term morbidity, which includes memory deficits and depression. Brain recovery from injury involves widespread cellular proliferation in the brain, including in the hippocampus, a region highly involved in memory function and mood regulation. The N-MethylD-Aspartate-type glutamate receptor plays an essential role regulating hippocampal cell proliferation, including adult neurogenesis, and is inhibited by several drugs, including ketamine. As ketamine is increasingly being utilized in the management of TBI, we sought to determine its effects on hippocampal cell proliferation and outcomes using a mouse model of TBI.
RESULTS
(A) Low-power images demonstrate CCI-induced cortical cavitation above the hippocampus at 2 and 6 weeks post-injury. (B) Ketamine administration did not alter the injury size at either 2 and 6 weeks post-injury..(C) CCI induced a decrease in c-fos activation (c-fos+, green) at 3 days after injury that was not affected by ketamine treatment. (D) Quantification of cfos expression demonstrated a significant decrease in CCI groups compared to Sham groups. (E) All mouse weights increased equally in the weeks following CCI or sham, and were not affected by ketamine treatment.
FUNDING •FAER Research Fellowship Grant (Austin Peters) •VHA CDA-2 award 005-10S (Eric Schnell) •VHA Merit Award I01-BX002949 (Eric Schnell) •OHSU Department of Anesthesiology & Perioperative Medicine •OCTRI HIP Grant TL1TR000129 (Austin Peters) •NINDS P30 grant (P30 NS061800; Aicher, PI)
ABSTRACT A
RESULTS
RESULTS
Ketamine Increases Hippocampal Cell Proliferation After CCI
Ketamine Inhibits Astrogliogenesis After CCI
Ketamine Ameliorates the CCI-Induced Impairment in Water Maze Reversal Learning
Vehicle
Ketamine
B
3 Days Post-CCI Sham + Vehicle
CCI + Vehicle
2 Weeks Post-CCI Sham + Vehicle
CCI + Vehicle
Sham
Sham + Ketamine
Sham + Ketamine
DAPI NeuN
C
DAPI BrdU
6 Weeks Post-CCI Sham + Vehicle
CCI + Vehicle
D
BrdU+ Cells
C DAPI GFAP Sham + Ketamine
CCI + Ketamine
(A) Representative images of Sham (non-injured) and CCI-injured coronal sections at 2 weeks post-injury. (B) Ketamine administration after CCI induced a robust increase in newly born (BrdU+, red) cells in the granule cell layer 3 days after injury. (C) Images of BrdU-stained dentate gyrus taken 6 weeks after CCI, demonstrating increased BrdU staining. (D) Quantification of BrdU+ labeled cells over time at the various time points evaluated in this study.
Ketamine Treatment Facilitates Rapid Transient Microgliogenesis After A B 3 Days Post-CCI CCI Sham + Vehicle
2 Weeks Post-CCI Sham + Vehicle
CCI + Vehicle
Sham + Ketamine
CCI + Ketamine
C
DAPI BrdU Iba1
Ketamine Does Not Modulate Other Indicators of Injury Severity Injury Sizes After CCI
Sham + Ketamine
CCI + Ketamine
CCI + Ketamine
B
6 WPI
IHC
C
C-fos Expression 3 Days Post-CCI Sham + Vehicle
CCI + Vehicle
Sham + Ketamine
CCI + Ketamine
D
E
DAPI cfos
(A) Representational images of the granule cell layer of the 4 treatment groups at 6 weeks after injury. Dcx+/BrdU+ co-labeling (red arrows) represents either continued mitotic activity of cells born after injury or persistent expression of the immature neuronal marker Dcx. (B) CCI decreased Dcx labeling across treatment groups with no change in ketamine vs. vehicle exposure. (C) Dcx+/BrdU+ co-labeled cells were not significantly increased between individual groups, but there was an overall significant increase Dcx+/BrdU+ co-labeling in all CCI groups versus Sham groups.
•Ketamine after TBI induces rapid early cellular proliferation (largely composed of microgliogenesis) and promotes survival of cells. •Ketamine transiently reduces astrogliogenesis and neurogenesis. •Ketamine rescues MWM reversal task performance after TBI. •TBI induces long term suppression of neurogenesis. •Improved MWM reversal may be related to the observed cellular responses, subtle anti-inflammatory changes not seen in this study, or off-target receptor activity by ketamine. Additional studies focusing on cellular mechanisms of ketamine and hippocampal stem cell responses to TBI are needed.
2 WPI
CCI + Vehicle
DAPI NeuN BrdU
Week 6
Week 4
Forced Swim Test Results
C
CCI + Ketamine
(A) Representative images of the GCL (DAPI, blue), microglia (Iba1+, white) and newly generated BrdU-labeled cells (green) at 3 days after intervention. Newly generated microglia (Iba1+/BrdU+) were greatly increased only in the Ketamine + CCI group at this time point. (B) Quantification of microgliogenesis 3 days after injury demonstrated a significant increase only in the Ketamine + CCI group. (C) At 6 weeks post-intervention, post-CCI-born microglia were significantly increased in both CCI groups versus their respective Sham groups.
CCI Delays the Maturation of Adult Born Neurons and Suppresses Hippocampal Neurogenesis Independent of Ketamine Behavioral Testing
IHC Week 2
Pump removal Day 7
BrdU BrdU vs IHC Day 2 Day 3
D
CONCLUSIONS
A
Intervention
CCI + Vehicle
(A) All groups successfully demonstrated preference for the target location in the Morris Water Maze (MWM), indicating grossly intact hippocampal function. (B) In the reversal task of the MWM, the CCI + Vehicle group failed to adapt to the new location, whereas the CCI + Ketamine group was able to achieve the task. (C) Characteristic tracings of each group from the reversal probe trials. (D) In the Forced Swim Test, ketamine administration did not affect the performance of CCI-induced mice.
B
(A) Representative images from each treatment group at 2 weeks post-injury. Immature neurons are stained with Dcx (white), along with BrdU-labeled cells (red) and DAPI (blue). Arrows indicate BrdU+/Dcx+ co-labeled cells. (B) Quantification of Dcx staining at 2 WPI demonstrated that ketamine administration suppressed neurogenesis after CCI. (C) Ketamine administration suppressed the number of neurons born 2-3 days after injury, as identified using the density of BrdU+/Dcx+ co-labaled cells.
Day 0
Sham + Vehicle
CCI + Vehicle
Post-Injury Ketamine Inhibits Neurogenesis 2 Weeks After CCI A
MWM Reversal Pathway Tracings
(A) Representative images of the four treatment groups at 2 weeks after intervention. The CCI + Vehicle group demonstrated a robust astrocytic response (GFAP+, red) not seen in the CCI + Ketamine group. (B) Quantification of astrogliogenesis 2 weeks post-injury demonstrated a significant increase in new astroglia (GFAP+/Dcx+ cells) in the Vehicle + CCI group that was blocked by ketamine.
DAPI BrdU Dcx
Mice were randomly assigned to receive CCI or Sham (no-injury), and subsequently implanted with an osmotic drug pump delivering ketamine or vehicle (saline). Dividing cells were labeled with BrdU at 2 and/or 3 days after the intervention. Immunohistochemical assays were employed to evaluate cellular responses in the granule cell layer of the hippocampus at 3 days, 2 weeks and 6 weeks after the intervention. Behavioral testing was conducted 4 weeks after the intervention.
Morris Water Maze Reversal Results
CCI + Ketamine
Sham + Ketamine
DESIGN
B
CCI + Ketamine
DAPI BrdU
Conclusions Ketamine alters cell proliferation after traumatic brain injury and suppresses constitutive and post-injury neurogenesis. Surprisingly, it improved behavioral outcomes specific to neurogenesis-related tasks, suggesting that the benefit of this treatment may have been due to ketamine-induced qualitative, and not quantitative effects on neurogenesis or due to changes in the glial modulation of neuronal circuit function.
Morris Water Maze (MWM) Results
B
Methods TBI was modeled in mice using controlled cortical impact (CCI) followed by a one-week continuous infusion of either vehicle or ketamine by osmotic pump. We utilized immunohistochemical and behavioral assays to evaluate cellular responses and recovery at different time points after injury. Results Ketamine treatment significantly increased hippocampal cell proliferation at early and late time points after CCI. Ketamine treatment after CCI also increased early microglial proliferation and reduced astrogliogenesis. Independent of injury, ketamine temporarily reduced neurogenesis. Ketamine administration rescued hippocampus dependent context-separation deficits of injured mice, as assayed by the Morris Water Maze reversal test.
A
A
2 WPI
Background Traumatic brain injury (TBI) is associated with long-term morbidity, which includes memory deficits and depression. Brain recovery from injury involves widespread cellular proliferation in the brain, including in the hippocampus, a region highly involved in memory function and mood regulation. The N-MethylD-Aspartate-type glutamate receptor plays an essential role regulating hippocampal cell proliferation, including adult neurogenesis, and is inhibited by several drugs, including ketamine. As ketamine is increasingly being utilized in the management of TBI, we sought to determine its effects on hippocampal cell proliferation and outcomes using a mouse model of TBI.
RESULTS
(A) Low-power images demonstrate CCI-induced cortical cavitation above the hippocampus at 2 and 6 weeks post-injury. (B) Ketamine administration did not alter the injury size at either 2 and 6 weeks post-injury..(C) CCI induced a decrease in c-fos activation (c-fos+, green) at 3 days after injury that was not affected by ketamine treatment. (D) Quantification of cfos expression demonstrated a significant decrease in CCI groups compared to Sham groups. (E) All mouse weights increased equally in the weeks following CCI or sham, and were not affected by ketamine treatment.
FUNDING •FAER Research Fellowship Grant (Austin Peters) •VHA CDA-2 award 005-10S (Eric Schnell) •VHA Merit Award I01-BX002949 (Eric Schnell) •OHSU Department of Anesthesiology & Perioperative Medicine •OCTRI HIP Grant TL1TR000129 (Austin Peters) •NINDS P30 grant (P30 NS061800; Aicher, PI)
