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. 2017 May 19;12(5):e0178013.
doi: 10.1371/journal.pone.0178013. eCollection 2017.

Therapeutic Benefits of Phosphodiesterase 4B Inhibition After Traumatic Brain Injury

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Free PMC article

Therapeutic Benefits of Phosphodiesterase 4B Inhibition After Traumatic Brain Injury

Nicole M Wilson et al. PLoS One. .
Free PMC article

Abstract

Traumatic brain injury (TBI) initiates a deleterious inflammatory response that exacerbates pathology and worsens outcome. This inflammatory response is partially mediated by a reduction in cAMP and a concomitant upregulation of cAMP-hydrolyzing phosphodiesterases (PDEs) acutely after TBI. The PDE4B subfamily, specifically PDE4B2, has been found to regulate cAMP in inflammatory cells, such as neutrophils, macrophages and microglia. To determine if PDE4B regulates inflammation and subsequent pathology after TBI, adult male Sprague Dawley rats received sham surgery or moderate parasagittal fluid-percussion brain injury (2 ± 0.2 atm) and were then treated with a PDE4B - selective inhibitor, A33, or vehicle for up to 3 days post-surgery. Treatment with A33 reduced markers of microglial activation and neutrophil infiltration at 3 and 24 hrs after TBI, respectively. A33 treatment also reduced cortical contusion volume at 3 days post-injury. To determine whether this treatment paradigm attenuated TBI-induced behavioral deficits, animals were evaluated over a period of 6 weeks after surgery for forelimb placement asymmetry, contextual fear conditioning, water maze performance and spatial working memory. A33 treatment significantly improved contextual fear conditioning and water maze retention at 24 hrs post-training. However, this treatment did not rescue sensorimotor or working memory deficits. At 2 months after surgery, atrophy and neuronal loss were measured. A33 treatment significantly reduced neuronal loss in the pericontusional cortex and hippocampal CA3 region. This treatment paradigm also reduced cortical, but not hippocampal, atrophy. Overall, these results suggest that acute PDE4B inhibition may be a viable treatment to reduce inflammation, pathology and memory deficits after TBI.

Conflict of interest statement

Competing Interests: NMW declares no competing financial interests. MEG, WDD, and CMA are co-inventors on USPTO 8,865,723 which claims therapeutic use of PDE4B inhibitors for treating brain injury. Tetra Discovery Partners (MEG) is the owner of this patent. WDD and CMA do not have equity stakes in the company and do not stand to receive royalties. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The compound A33 is public domain and the chemical structure is available online for synthesis by interested parties.

Figures

Fig 1
Fig 1. PDE4B2 is expressed in microglia and infiltrating myeloid-lineage cells in the injured cortex at 24 hrs post-TBI.
Representative scatter plots from the ipsilateral parietal cortex of (A) sham and (B) brain-injured animals. (C) Quantification of PDE4B2+ inflammatory cells in the cortex at 24 hrs post-surgery. Mean ± SEM, n = 8-10/group, *p<0.05, **p<0.01, ***p<0.001 vs. Sham, Student’s unpaired t-test. ND = not detectable.
Fig 2
Fig 2. Distribution of A33 to the brain and plasma and effects on cAMP levels.
(A) A33 levels were measured in the plasma and brain at 1 hr after A33 administration (0.3 mg/kg, i.p.). A33 levels were significantly higher in the brains of TBI animals as compared to sham animals (main effect of surgery: F(1,8) = 11.986, p = 0.009 for brain A33 levels). Mean ± SEM, n = 3/group, two-way ANOVA with post-hoc Student-Newman-Keuls (brain), Student’s unpaired t-test (plasma). (B) Total cAMP levels were significantly decreased in the ipsilateral parietal cortex of vehicle- and A33-treated TBI animals as compared to sham animals. A33 treatment did not increase total cAMP in the cortex at 6 hrs post-injury as compared to vehicle-treated TBI animals. Mean ± SEM, n = 9-12/group, **p<0.01, ***p<0.001 vs. Sham, one-way ANOVA with post-hoc Student-Newman-Keuls.
Fig 3
Fig 3. Neutrophil accumulation is reduced with A33 treatment at 24 hrs after TBI.
Representative CD45/CD11b and CD45/RP-1 scatter plots from the ipsilateral parietal cortex of (A) vehicle-treated and (B) A33-treated animals at 3 hrs post-injury. CD45/RP-1 scatter plots were gated on total CD11b+ cells. (C) Quantification of total CD11b+ cells, microglia, infiltrating CD11b+ cells and neutrophils in vehicle-treated and A33-treated TBI animals at 3 hrs post-injury. (D) Percentage of infiltrating CD11b+ cells that are neutrophils (CD45high, CD11b+, RP-1+). Representative CD45/CD11b and CD45/RP-1 scatter plots at 24 hrs post-injury in (E) vehicle-treated and (F) A33-treated animals. (G) Quantification of total CD11b+ cells, microglia, infiltrating CD11b+ cells and neutrophils. (H) Percentage of infiltrating CD11b+ cells that are neutrophils at 24 hrs after TBI. Mean ± SEM, n = 10/group (3 hrs), n = 5/group (24 hrs), **p<0.01 vs. TBI+Vehicle, Student’s unpaired t-test. (RP-1 = rat neutrophil marker).
Fig 4
Fig 4. A33 treatment increased Arg1-expressing microglia and myeloid-lineage cells at 3 hrs after TBI.
Representative histogram overlay of iNOS+ and Arg1+ (A) infiltrating CD11b+ cells and (B) microglia at 3 hrs post-injury. Quantification of iNOS and Arg1-expressing (C) infiltrating CD11b+ cells and (D) microglia. Representative histogram overlay of iNOS+ and Arg1+ (E) infiltrating CD11b+ cells and (F) microglia at 24 hrs post-injury. Quantification of iNOS and Arg1-expressing (G) infiltrating CD11b+ cells and (H) microglia at 24 hrs after TBI. Mean ± SEM, n = 10/group (3 hrs), n = 5/group (24 hrs), *p<0.05, **p<0.01 vs. TBI+Vehicle, Student’s unpaired t-test. (iNOS = inducible nitric oxide synthase, Arg1 = arginase 1).
Fig 5
Fig 5. Acute PDE4B inhibition reduced cortical contusion volume at 3 days after TBI.
Representative images of the ipsilateral parietal cortex stained with H&E from (A,C) vehicle-treated and (B,D) A33-treated animals at 3 days post-injury. Representative bregma level -6.3 mm. Scale bars (A,B) 1 mm and (C,D) 500 μm. (E) Quantification of contusion volume. Mean ± SEM, n = 10/group, ***p<0.001 vs. TBI+Vehicle, Student’s unpaired t-test.
Fig 6
Fig 6. Acute PDE4B inhibition did not rescue sensorimotor deficits at 1 week after TBI.
Animals were evaluated for spontaneous forelimb use asymmetry at 1 week after surgery using the cylinder task. Mean ± SEM, n = 12-14/group, *p<0.05 vs. Sham, one-way ANOVA with post-hoc Student-Newman-Keuls.
Fig 7
Fig 7. Acute PDE4B inhibition reduced contextual fear conditioning deficits at 2 weeks after TBI.
(A) At 24 hrs and 1 month after training, animals were evaluated for contextual fear conditioning. At both 24 hrs and 1 month after training, vehicle-treated TBI animals froze significantly less than sham animals. A33-treated TBI animals froze significantly more than vehicle-treated TBI at 24 hrs, but not at 1 month, after training. A main effect of treatment (F(2, 72) = 6.996, p = 0.003), a main effect of trial (F(2, 72) = 169.111, p<0.001) and a significant interaction of treatment x trial (F(4, 72) = 4.931, p = 0.001) were observed. *p<0.05, ***p<0.001 vs. Sham Day 1, **p<0.01 vs. Sham Day 30, #p<0.001 vs. Training, +p<0.01 vs. TBI+Vehicle Day 1, repeated-measures two-way ANOVA with post-hoc Student-Newman-Keuls. (B) Shock threshold was similar between all treatment groups. Mean ± SEM, n = 12-14/group, one-way ANOVA with post-hoc Student-Newman-Keuls.
Fig 8
Fig 8. Acute PDE4B inhibition attenuated spatial memory deficits in the water maze task at 3 weeks post-TBI.
During training, (A) escape latency and (B) path length were significantly increased in the vehicle-treated TBI animals as compared to sham (main effect of treatment: F(2,111) = 5.446, p = 0.008 and F(2,111) = 5.292, p = 0.010 for escape latency and path length, respectively), repeated-measures two-way ANOVA with post-hoc Student-Newman-Keuls. (C) During the probe trial, the percentage of time spent in the target quadrant (TQ) was significantly decreased in the vehicle-treated TBI animals as compared to sham and A33-treated TBI animals. (D) There was no difference in swim velocity between any of the treatment groups. Mean ± SEM, n = 12-14/group, *p<0.05, **p<0.01 vs. TBI+Vehicle, one-way ANOVA with post-hoc Student-Newman-Keuls.
Fig 9
Fig 9. Acute PDE4B inhibition did not rescue spatial working memory deficits at 1 month post-TBI.
(A) Path length from location and match trials. (B) Path length analysis of match trial. Both vehicle and A33-treated TBI animals had significantly impaired working memory, as measured by an increase in path length in the match trial as compared to sham animals. Mean ± SEM, n = 12-14/group, *p<0.05, **p<0.01 vs. Sham, one-way ANOVA with post-hoc Student-Newman-Keuls.
Fig 10
Fig 10. Acute PDE4B inhibition reduced cortical atrophy at 2 months post-injury.
Representative images of the ipsilateral parietal cortex and hippocampus of (A) sham, (B) vehicle-treated and (C) A33-treated TBI animals stained with H&E plus Luxol fast blue. Representative images at -6.3 mm bregma, scale bar 500 μm. (D) Quantification of % atrophy. Both vehicle and A33-treated TBI animals had significantly increased cortical and hippocampal atrophy as compared to sham animals. A33-treated TBI animals had reduced cortical, but not hippocampal, atrophy as compared to vehicle-treated TBI animals. Mean ± SEM, n = 11-14/group, **p<0.01, ***p<0.001 vs. Sham, #p<0.001 vs. TBI+Vehicle, one-way ANOVA with post-hoc Student-Newman-Keuls.
Fig 11
Fig 11. A33 treatment reduced neuronal loss in the pericontusional cortex at 2 months post-injury.
Representative images of the ipsilateral parietal cortex of (A) sham, (B) vehicle-treated and (C) A33-treated TBI animals, immunolabeled for mature neurons using NeuN. Representative images at -5.3 mm bregma, scale bar 250 μm. (D) Quantification of NeuN+ cells in the ipsilateral and contralateral parietal cortex. The number of NeuN+ cells were significantly reduced in the ipsilateral parietal cortex in both vehicle and A33-treated TBI animals as compared to sham animals. A33 treatment rescued neuronal loss in the pericontusional cortex as compared to vehicle-treated TBI animals. (main effect of treatment: F(2, 64) = 19.21, p<0.0001; main effect of region (ipsi vs. contra): F(1, 64) = 93.68, p<0.0001; interaction of treatment x region: F(2, 64) = 43.89, p<0.0001.) Mean ± SEM, n = 10-13/group, ***p<0.001 vs. Ipsi/Contra Sham, #p<0.001 vs. Ipsi TBI+Vehicle, +p<0.001 vs. Ipsi TBI+A33, two-way ANOVA with post-hoc Student-Newman-Keuls.
Fig 12
Fig 12. Treatment with a PDE4B inhibitor reduced neuronal loss in the hippocampal CA3 region at 2 months post-injury.
Representative images of the ipsilateral CA3 region of the hippocampus immunolabeled for mature neurons using NeuN in (A) sham, (B) vehicle-treated and (C) A33-treated TBI animals. Representative images at -5.3 mm bregma, scale bar 250 μm. (D) Quantification of NeuN+ cells in the ipsilateral and contralateral CA3 region of the hippocampus. Vehicle and A33-treated TBI animals had significantly reduced NeuN+ cells in the ipsilateral hippocampal CA3 region as compared to sham animals. A33 treatment partially rescued neuronal loss in the ipsilateral hippocampal CA3 region as compared to vehicle-treated TBI animals. (main effect of treatment: F(2, 64) = 8.977, p = 0.0004; main effect of region (ipsi vs. contra): F(1, 64) = 18.45, p<0.0001; interaction of treatment x region: F(2, 64) = 13.38, p<0.0001.) Mean ± SEM, n = 10-13/group, **p<0.01, ***p<0.001 vs. Ipsi/Contra Sham, #p<0.01, ##p<0.001 vs. Ipsi TBI+Vehicle, two-way ANOVA with post-hoc Student-Newman-Keuls.

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