Drabek T, Janata A, Jackson EK, End B, Stezoski J, Vagni VA, Janesko-Feldman K, Wilson CD, van Rooijen N, Tisherman SA, Kochanek PM.
Microglial depletion using intrahippocampal injection of liposome-encapsulated clodronate in prolonged hypothermic cardiac arrest in rats.
Resuscitation. 2012 Apr;83(4):517–26.
[toggle_content title=”Abstract”] Trauma patients who suffer cardiac arrest (CA) from exsanguination rarely survive. Emergency preservation and resuscitation using hypothermia was developed to buy time for resuscitative surgery and delayed resuscitation with cardiopulmonary bypass (CPB), but intact survival is limited by neuronal death associated with microglial proliferation and activation. Pharmacological modulation of microglia may improve outcome following CA. Systemic injection of liposome-encapsulated clodronate (LEC) depletes macrophages. To test the hypothesis that intrahippocampal injection of LEC would attenuate local microglial proliferation after CA in rats, we administered LEC or PBS into the right or left hippocampus, respectively. After rapid exsanguination and 6 min no-flow, hypothermia was induced by ice-cold (IC) or room-temperature (RT) flush. Total duration of CA was 20 min. Pre-treatment (IC, RTpre) and post-treatment (RTpost) groups were studied, along with shams (cannulation only) and CPB controls. On day 7, shams and CPB groups showed neither neuronal death nor microglial activation. In contrast, the number of microglia in hippocampus in each individual group (IC, RTpre, RTpost) was decreased with LEC vs. PBS by ∼34–46% (P < 0.05). Microglial proliferation was attenuated in the IC vs. RT groups (P < 0.05). Neuronal death did not differ between hemispheres or IC vs. RT groups. Thus, intrahippocampal injection of LEC attenuated microglial proliferation by ∼40%, but did not alter neuronal death. This suggests that microglia may not play a pivotal role in mediating neuronal death in prolonged hypothermic CA. This novel strategy provides us with a tool to study the specific effects of microglia in hypothermic CA. [/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]
Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
5 mg/ml 20.4 mg/ml EPC/Chol/Aminophenyl mannose 69/21/10 MLV PBS
[/custom_table] [/toggle_content] [toggle_content title=”Animals and Dosing”] [custom_table]
Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Systemic Dosing? Systemic Results
Sprague Dawley rats, male, 300-350 g 5 µl stereotaxic inj/hippocampus Iba-1+ microglia no no change in peripheral moncytes or PMN detected
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Detailed description of stereotaxic injection of 5 µl of liposomal clodronate (R hemisphere) and 5 µl control liposomes (L hemisphere) over 10 min.
  2. Needle was left in place for 3 min. post-injection and withdrawn at 1 mm/min.
  3. To assess potential increase in intracranial pressure (ICP) due to injection, 10 µl liposomal clodronate was injected in control animals and ICP monitored.
  4. Liposome treatment occured either 24 hr prior to or 24 hr after exsanguination and resusitation experimental protocol; animals sacrificed 7 days post-exsanguination protocol.
[/toggle_content] [toggle_content title=”Results”]
  1. ICP did not change significantly during injection of 10 µl of liposomal clodronate.
  2. Iba-1 detects activated microglia which were reduced overall by 37-42% (immunohistology) by liposomal clodronate.
  3. The post-trauma treatment appeared to reduce the microglia by 50% compared to the pre-trauma treatment in both liposome treatment groups.
  4. While neuronal death overall was not affected, again the neuronal death was lower in both liposome treatment groups when comparing pre- and post-trauma treatment groups.
  5. The authors attribute these results (points 3 and 4) to the effect of the pre- and post-trauma treatment regimen which would be supported by the data if the liposomal PBS control group values did not change with treatment regimen. However, since the effect is observed in both liposomal PBS and liposomal clodronate groups, it seems that the effect is a result of the presence of liposomes (with or without clodronate). Therefore, it is critical to compare buffer-injected controls to both liposomal clodronate and liposomal PBS treated animals in determining the effect of treatment regimen on microglial depletion and neuronal death. If we speculatively assume that buffer treatment will have no effect, then liposomes (with or without clodronate) themselves appear to reduce neuronal death depending on treatment regimen. There is even a slight suggestion that liposomal PBS is more effective than liposomal clodronate (31% vs 19% reduction in neuronal death).
  6. These observations also hold true for the microglial depletion data except that liposomal clodronate does reduce the number of activated microglia as the authors conclude. However, the presence of liposomes, even without clodronate, reduces the number of active microglia by at least half when dosed 24 hr post-trauma rather than 24 hr pre-trauma. Although the specific reduction in activated microglia by liposomal clodronate does not translate to neuronal sparing, the overall reduction in activated microglia when either liposomal PBS or liposomal clodronate is dosed 24 hr post-trauma does correlate to neuronal sparing.
  7. The authors viewed this study as a proof-of-concept trial and acknowledged several other parameters, including determining the optimal liposomal clodronate dose and treatment regimen, and evaluating effects on other inflammatory cell types (i.e. PMN). However, the observation that liposomes, even without clodronate, attenuates microglial activation/proliferation and neuronal death by a mechanism apparently unrelated to macrophage depletion points in a different direction for neuroprotection post severe CV trauma— can liposomes, perhaps incombination with other anti-inflammatory agents, ameliorate (or prevent) neuronal loss post-CV-trauma?