Early low-anticoagulant desulfated heparin after traumatic brain injury: Reduced brain edema and leukocyte mobilization is associated with improved watermaze learning ability weeks after injury

Abstract

Background: Unfractionated heparin administered immediately after traumatic brain injury (TBI) reduces brain leukocyte (LEU) accumulation, and enhances early cognitive recovery, but may increase bleeding after injury. It is unknown how non-anticoagulant heparins, such as 2,3-O desulfated heparin (ODSH), impact post-TBI cerebral inflammation and long-term recovery. We hypothesized that ODSH after TBI reduces LEU-mediated brain inflammation and improves long-term neurologic recovery.

Methods: CD1 male mice (n = 66) underwent either TBI (controlled cortical impact [CCI]) or sham craniotomy. 2,3-O desulfated heparin (25 mg/kg [25ODSH] or 50 mg/kg [50ODSH]) or saline was administered for 48 hours after TBI in 46 animals. At 48 hours, intravital microscopy visualized rolling LEUs and fluorescent albumin leakage in the pial circulation, and the Garcia Neurologic Test assessed neurologic function. Brain edema (wet/dry ratio) was evaluated post mortem. In a separate group of animals (n = 20), learning/memory ability (% time swimming in the Probe platform quadrant) was assessed by the Morris Water Maze 17 days after TBI. Analysis of variance with Bonferroni correction determined significance (p < 0.05).

Results: Compared with CCI (LEU rolling: 32.3 ± 13.7 LEUs/100 μm per minute, cerebrovascular albumin leakage: 57.4 ± 5.6%), both ODSH doses reduced post-TBI pial LEU rolling (25ODSH: 18.5 ± 9.2 LEUs/100 μm per minute, p = 0.036; 50ODSH: 7.8 ± 3.9 LEUs/100 μm per minute, p < 0.001) and cerebrovascular albumin leakage (25ODSH: 37.9 ± 11.7%, p = 0.001, 50ODSH: 32.3 ± 8.7%, p < 0.001). 50ODSH also reduced injured cerebral hemisphere edema (77.7 ± 0.4%) vs. CCI (78.7 ± 0.4 %, p = 0.003). Compared with CCI, both ODSH doses improved Garcia Neurologic Test at 48 hours. Learning/memory ability (% time swimming in target quadrant) was lowest in CCI (5.9 ± 6.4%) and significantly improved in the 25ODSH group (27.5 ± 8.2%, p = 0.025).

Conclusion: 2,3-O desulfated heparin after TBI reduces cerebral LEU recruitment, microvascular permeability and edema. 2,3-O desulfated heparin may also improve acute neurologic recovery leading to improved learning/memory ability weeks after injury.

Figure 1.

Experimental design and procedures. (A) Timeline of SERIES 1 experimental protocol. (B) Timeline of SERIES 2 experimental protocol. IVM, intravital microscopy; NS, normal saline.

Figure 2.

In vivo LEU/EC interactions and microvascular permeability. (A) Representative image showing LEU interacting with endothelium. White arrows indicate fluorescently labeled LEU rolling on endothelium. (B) Representative image showing FITC-albumin leakage in the cerebral microcirculation. The microvascular permeability index is expressed as the ratio of mean fluorescence of three separate locations outside the vessel wall (perivenular intensity, IP) to mean fluorescence of three separate locations within the venule (venular intensity, IV). (C) LEU rolling and FITC-albumin leakage in the pial penumbral microcirculation 48 hours after CCI. Compared with positive controls (CCI), both ODSH regimens reduced post-TBI LEU rolling and cerebrovascular albumin leakage. *p < 0.05, **p < 0.01 vs. CCI, ^#p < 0.05, ^##p < 0.01 vs. 25ODSH.

Figure 3.

Tissue edema. (A) High-dose ODSH (50ODSH) reduced brain water content in the injured (ipsilateral) cerebral hemisphere to levels nearing negative controls (noCCI). **p < 0.01 vs. CCI in the ipsilateral hemisphere. Contralateral cerebral edema was similar in all groups. (B) There were no statistical differences in lung water content among groups.

Figure 4.

Neurologic function was normal in negative controls at both 24-hour and 48-hour timeframes. Compared to positive CCI controls, both ODSH treatments improved GNT at both time frames after injury. *p < 0.05, **p < 0.01 vs. CCI, ^##p < 0.01 vs. 25ODSH, ^{◊ ◊} p < 0.01 vs. 50ODSH.

Figure 5.

Uninjured animals recovered normal swimming velocity by day 15. Only 50ODSH injured animals recovered normal swimming velocity as compared with untreated injured (CCI) animals by day 16 and 17. *p < 0.05, **p < 0.01 vs. CCI, #p < 0.05, ##p < 0.01 vs. 25ODSH.

Figure 6.

Spacial learning and memory. (A) % animal swimming time in the target quadrant where platform was previously (probe trial). Compared to positive CCI controls, noCCI and 25ODSH animals spent significantly more time in the target quadrant. (B) Representative image sequence of the geographical position of a CCI animal in the 60-s observation period. Each dot is the position of the animal at 33-ms intervals. (C) Representative image sequence of the geographical position of a noCCI animal in the 60-second observation period.