Post-Injury Neuroprotective Effects of the Thalidomide Analog 3,6'-Dithiothalidomide on Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) is a major cause of mortality and disability worldwide. Long-term deficits after TBI arise not only from the direct effects of the injury but also from ongoing processes such as neuronal excitotoxicity, inflammation, oxidative stress and apoptosis. Tumor necrosis factor-α (TNF-α) is known to contribute to these processes. We have previously shown that 3,6'-dithiothalidomide (3,6'-DT), a thalidomide analog that is more potent than thalidomide with similar brain penetration, selectively inhibits the synthesis of TNF-α in cultured cells and reverses behavioral impairments induced by mild TBI in mice. In the present study, we further explored the therapeutic potential of 3,6'-DT in an animal model of moderate TBI using Sprague-Dawley rats subjected to controlled cortical impact. A single dose of 3,6'-DT (28 mg/kg, i.p.) at 5 h after TBI significantly reduced contusion volume, neuronal degeneration, neuronal apoptosis and neurological deficits at 24 h post-injury. Expression of pro-inflammatory cytokines in the contusion regions were also suppressed at the transcription and translation level by 3,6'-DT. Notably, neuronal oxidative stress was also suppressed by 3,6'-DT. We conclude that 3,6'-DT may represent a potential therapy to ameliorate TBI-induced functional deficits.

Keywords: 3,6′-dithiothalidomide; neurodegeneration; neuroinflammation; neurological deficits; oxidative stress; traumatic brain injury.

Figure 1

3,6′-dithiothalidomide (3,6′-DT) significantly attenuates the generation of TNF-α and nitrite, and the induction of iNOS protein in LPS activated RAW 264.7 cells. Media nitrite levels were significantly reduced at all 3,6′-DT concentrations evaluated. Media TNF-α protein levels were significantly reduced at 30 µM 3,6′-DT. Cellular levels of iNOS were significantly decreased in drug treated cells at a concentration of 10 and 30 µM. Only cells treated with 3,6′-DT at 60 µM indicated any evidence of cell toxicity, as determined by elevations in the absorbance of culture media observed from the lactate dehydrogenase assay (LDH). Drug effects on TNF-α, nitrite and iNOS at concentrations of 1 to 30 µM, were due to selective anti-inflammatory actions of the drug. Values are expressed as a percent change from drug vehicle control levels, the data are presented as mean ± S.E.M (n = 3 to 4 culture wells per group). * p < 0.05 and *** p < 0.001 compared with the respective control (CNT) group to which vehicle alone was added.

Figure 2

Post-injury administration of 3,6′-DT at 5 h after Traumatic brain injury (TBI) significantly reduced contusion volume evaluated at 24 h. (A) Representative cresyl violet stained coronal brain sections of TBI-induced cavity in Sham, TBI-Vehicle (TBI + Veh), and 3,6′-DT-treated TBI rats (TBI + 3,6′-DT) at 24 h post-TBI. (B) The TBI-induced contusion volume evaluated at 24 h was significantly reduced by 3,6′-DT treatment. Data are presented as mean ± S.E.M. (n = 5 in each group). * p < 0.05 and *** p < 0.001 compared with the Sham group. ^# p < 0.01 compared with the TBI + Veh group.

Figure 3

TBI induces neuron degeneration within the contusion regions, and treatment with 3,6′-DT reduced the number of TBI-induced degenerating neurons. (A) Representative HE-stained coronal brain section from Sham that shows the area of evaluation. (B) Representative photomicrographs showing the presence of fluoro-jade C (FJC)-staining at 24 h in different groups. (C) Quantitative comparison of mean densities of FJC-positive cells in the cortical contusion area at 24 h post-injury. Data are presented as mean ± S.E.M. (n = 5 in each group). *** p < 0.001 compared with the Sham group. Scale bar = 100 µm.

Figure 4

3,6′-DT administered at 5 h post-injury improved functional outcomes as revealed by behavioral evaluation at 24 h after TBI. (A) Motor asymmetry measured by the elevated body swing test (EBST). (B) Beam walk latency before and after TBI challenge. (C) Functional deficits measured by the mNSS score, and (D) adhesive removal latency from the left (L) and right (R) forepaw. Data are presented as mean ± S.E.M. (n = 5 in each group). *** p < 0.001 compared with Sham group; ^# p < 0.05, ^### p < 0.001 versus TBI + Veh group; ⁺⁺⁺ p < 0.001 compared with the value of left side.

Figure 5

Post-injury administration of 3,6′-DT at 5 h after TBI significantly decreased apoptotic neurons in the cortical contusion regions at 24 h. (A) The representative HE-stained coronal section showing the area as indicated by the black square box to compare the fluorescent signals across groups of rats. (B) The immunofluorescence of Annexin V and NeuN in cortical brain tissue. The Annexin V immunoreactivity is shown in green, and NeuN (a marker for neurons) is shown in red. The yellow color indicates colocalization. (C) The number of Annexin V/NeuN positive cells was elevated when evaluated at 24 h after TBI. Treatment with 3,6′-DT significantly decreased the number of apoptotic neurons compared with TBI + Veh animals. Data are presented as mean ± S.E.M. (n = 5 in each group). *** p < 0.001 compared with the Sham group. ^# p < 0.05 compared with the TBI + Veh group. Scale bar = 100 μm.

Figure 6

TBI induces microglia activation within the contusion regions, and treatment with 3,6′-DT significantly suppressed microglia activation. (A) A low power image of brain section immune-histochemically stained with Iba-1 antibody in cortical regions from a TBI animal showing the contusion site and area of observation (Black square). (B) Representative photomicrographs showing the Iba-1-positive cells with amoeboid (activated) or ramified (resting) phenotypes in cortical regions from various animal groups at 24 h after TBI. (C) Quantitative comparison of mean densities of Iba-1-positive cells in the cortical contusion area at 24 h post-injury. Microglial activation after TBI, as observed by the increased number and change of their morphologic phenotypes (from ramified to amoeboid), was reduced by 3,6′-DT treatment. Mean ± S.E.M. (n = 5 in each group). *** p < 0.001 compared with the Sham group. ^### p < 0.001 compared with the TBI + Veh group.

Figure 7

Treatment with 3,6′-DT at 5 h after TBI reduced injury-induced mRNA expression of cytokines at 8 h after TBI in the ipsilateral hemisphere cortex. TBI-induced elevations in mRNA levels of (A) TNF-α, (B) IL-1β and (C) IL-6. Data are presented as mean ± S.E.M. (n = 5 in each group). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the Sham group. ^### p < 0.001, compared with the TBI + Veh group.

Figure 8

Post-injury administration of 3,6′-DT at 5 h after TBI significantly reduced the elevated tissue levels of inflammatory cytokine proteins measured at 8 h. (A) TNF-α, (B) IL-1β, and (C) IL-6 protein levels in TBI + Veh rats were significantly elevated in the ipsilateral cortex at 8 h after injury. Administration of 3,6′-DT mitigated the rise in these three cytokine levels, which reached statistical significance for TNF-α, and IL-1β, but not IL-6. Data are presented as mean ± S.E.M. (n = 5 in each group). *** p < 0.001 compared with the Sham group. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 compared with the TBI + Veh group.

Figure 9

Administration of 3,6′-DT at 5 h post-injury reduced injury-induced lipid peroxidation product in the cortical contusion regions at 24 h. (A) The representative HE-stained coronal section showing the area as indicated by the black square box to compare the fluorescent signals across 3 groups of rats. (B) The immunofluorescence of 4-hydroxynonenal, 4-HNE (lipid peroxidation product), and NeuN in cortical brain tissue. The 4-HNE immunoreactivity is shown in green, and NeuN (a marker for neurons) is shown in red. The yellow cells indicate colocalization. (C) There was a significant CCI TBI-induced elevation in the number of 4-HNE positive neurons that was significantly ameliorated in the TBI + 3,6′-DT group. Data are presented as mean ± S.E.M. (n = 5 in each group). ** p < 0.01, *** p < 0.001 compared with the Sham group. ^# p < 0.05 compared with the TBI + Veh group. Scale bar = 100 µm.

Figure 10

Administration of 3,6′-DT at 5 h post-injury reduced injury-induced tyrosine nitration product in the cortical contusion regions at 24 h. (A) The representative HE-stained coronal section indicates the area used to compare the fluorescent cell observations in different animal treatment groups. (B) The immunofluorescence of 3-nitrotyrosine, 3-NT (tyrosine nitration product mediated by reactive nitrogen species), and NeuN in cortical brain tissue. The 3-NT immunoreactivity is shown in green, and NeuN is shown in red. The yellow color indicates colocalization. (B) Compared to TBI + Veh animals there was a significant decrease in the number of 3-NT/NeuN positive cells in the TBI + 3,6′-DT group. Data are presented as mean ± S.E.M. (n = 5 in each group). *** p < 0.001 compared with the Sham group. ^# p < 0.05 compared with the TBI + Veh group. Scale bar = 100 µm.