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Amphiphilic, tri-block copolymers provide potent membrane-targeted neuroprotection¹

JEREMY D. MARKS^*2, CHIEN-YUAN PAN, TREVOR BUSHELL, WILLIAM CROMIE and RAPHAEL C. LEE

Departments of
^* Pediatrics,
Physiological and Pharmacological Sciences, and
Surgery, University of Chicago, Chicago, Illinois 60637, USA

²Correspondence: Department of Pediatrics, MC 6060, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637 USA. E-mail: j-marks1{at}uchicago.edu

SPECIFIC AIMS

The aim of our study was to investigate the potential neuroprotective effects of Poloxamer 188 (P188), one of a class of amphiphilic tri-block copolymers, on neuronal survival after stimuli resulting in neuronal necrosis. We hypothesized that the loss of plasma membrane integrity, which is the hallmark of neuronal necrosis, represents a pivotal event in the pathway of necrotic injury and that molecules restoring membrane integrity constitute a potentially important mode of neuroprotection.

PRINCIPAL FINDINGS

1. Poloxamer protects cultured neurons from NMDA
Embryonic (E17) rat hippocampal pyramidal neurons were exposed at 12 days in vitro to N-methyl-D-aspartate (NMDA, 300 µM) in HEPES-buffered saline for 15 min and survival was measured 48 h after exposure. NMDA exposure significantly reduced neuronal survival (33.8%±0.80% SEM NMDA vs. 71.05%±1.04% SEM vehicle; Fig. 1 ). Remarkably, addition of P188 to the culture medium after incubation in NMDA significantly increased 48 h survival to 73.3% ±0.66 SEM (P<.0001), slightly greater than vehicle alone, reducing NMDA-induced mortality to zero (Fig. 1) .

View larger version (74K): [in this window] [in a new window]   Figure 1. Poloxamer 188 protects neurons from stimuli-inducing necrosis in vitro. a) Representative fields of hippocampal neurons 48 h after 15 min exposure to buffer (left), NMDA (300 µm) alone (center), or NMDA followed by P188 (100 µM) in the culture media (right). Living neurons are stained with calcein (green) and dead neurons have nuclei stained with propidium iodide (red). b) Neuronal survival 48 h after intense excitotoxic (NMDA, kainate) and oxidative (menadione, tert-butyl-hydroperoxide) insults resulting in necrosis and staurosporine. Hippocampal neurons were used for all stimuli except kainate (100 µM), when cultured embryonic cerebellar Purkinje neurons were used. Bars represent sum of 6 coverslips per condition per toxin. Error bars are SEM. Statistical significance between groups (crosses: toxin treatment vs. control; asterisks: toxin with P188 vs. toxin alone) was determined with the likelihood ratio test (with correction for multiple comparisons) after overall significance by logistic regression. c) Survival of hippocampal neurons after NMDA (300 µM) and different concentrations of P188. Bars represent sum of 6 coverslips per condition per P188 concentration; error bars represent the standard error of the mean. Sham represents 15 min incubation in HEPES-buffered saline. Statistical significance between groups (crosses: toxin treatment vs. control; asterisks: toxin with P188 vs. toxin alone) determined as in panel b above. d) Survival of hippocampal neurons after NMDA and increasing delays between NMDA and addition of P188 to the cultures. Purkinje neurons were used. Bars represent sum of 6 coverslips per condition per toxin. Error bars are SEM. Statistical significance between groups (crosses: toxin treatment vs. control; asterisks: toxin with P188 vs. toxin alone) determined as in panel b above. e) Changes in [Ca2+]i, reported by Fura-2, of a single hippocampal neuron 5 days after exposure to NMDA (300 µm). After NMDA, P188 (30 µM) was added to the culture medium. High K represents exposure to KCl (60 mM). NMDA (2nd bar) is 300 µm.

2. Poloxamer protects cultured neurons from a variety of necrosis-inducing stimuli
To assess whether P188 protected neurons from non-NMDA receptor activation-induced injury, P188 was applied to hippocampal cultures after severe oxidative injury, with menadione (30 µM) or tert-butyl-hydroperoxide (100 µM). P188 effects on survival after kainate-induced injury were assessed by exposing cultured embryonic Purkinje neurons at 12 days in vitro to kainate (100 µM for 15 min). After exposure, 48 h survival fell from >70% to 30–50%, depending on the toxin (Fig. 1) . P188 (100 µM) added to the culture medium after toxin exposure significantly increased 48 h survival after all stimuli: survival approached control levels (P<.001 for each toxin; Fig. 1 ).

3. Poloxamer does not protect neurons from stimuli-inducing apoptosis
We assessed whether P188 protected neurons after induction of apoptosis, a process in which the plasma membrane remains intact. We measured P188 effects on hippocampal neuron survival 48 h after a 15 min incubation in staurosporine (200 nM). P188 increased 48 h survival after staurosporine only slightly (27.59± 1.3% SEM staurosporine vs. 37.75 ±2.08% SEM staurosporine, followed by P188; Fig. 1 ). This increase, although statistically significant (P<.01), was far less than the neuroprotection seen in the other models studied.

4. Poloxamer does not alter NMDA receptor currents or activation-induced [Ca²⁺]_i increases
To assess whether P188 reduces NMDA currents, we made whole-cell patch clamp recordings of NMDA-induced currents in embryonic hippocampal neurons in the absence and presence of P188 (100 µM). Peak NMDA-induced inward currents in the presence and absence of P188 were not significantly different (control 390 ±49 pA, P188 356 ±38 pA, n=5; P=0.5). We also measured NMDA effects on [Ca²⁺]_i in the absence and presence of P188 (100 µM), using time-lapse imaging of neurons loaded with the low-affinity Ca²⁺ reporter Fura-4F (K_d for Ca²⁺ 700 nM). Mean peak somal [Ca²⁺]_i during a 10 s exposure to NMDA (300 µM) did not differ significantly between the presence and absence of P188 (control 327 ±30 nM vs. P188 384 ±37 nM, n=13).

5. Neurons rescued by P188 demonstrate intact function
We assessed whether membrane receptor and intracellular functions are preserved in neurons rescued from NMDA by P188 treatment. Using Ca²⁺ imaging, we found that neurons exposed to intense NMDA receptor stimulation, followed by 48 h incubation in P188 (100 µM), produced a homogeneous population of neurons whose mean somal [Ca²⁺]_i at baseline was no different from baseline [Ca²⁺]_i of neurons not exposed to NMDA (70.6 ±4.3 nM SEM). Brief depolarization with 60 mM KCl abruptly increased mean somal [Ca²⁺]_i to 675 ± 119 nM SEM (Fig. 1) . Brief stimulation with NMDA (300 µM) increased mean somal [Ca²⁺]_i to 534 ± 99 nM SEM. After removal of each stimulus, [Ca²⁺]_i returned rapidly to baseline values. These results indicate that after P188-mediated rescue of neurons from NMDA injury: 1) [Ca²⁺]_i homeostatic mechanisms are intact; 2) voltage-gated Ca²⁺ channels and NMDA receptor-coupled Ca²⁺ channels function normally; and 3) after an intracellular Ca²⁺ load, [Ca²⁺]_i is appropriately decreased to baseline values.

6. Poloxamer inserts into the plasma membrane
To obtain direct evidence of insertion of P188 into the plasma membrane of intact cells, we measured P188-induced changes in cell surface area of bovine adrenal chromaffin cells using continuous measurements of whole cell capacitance. Mean capacitance of chromaffin cells at baseline was 5.1±0.28 pF (SEM, n=8). Perfusion of P188 (100 µM) increased cell capacitance in all cells for the duration of the perfusion (mean peak increase 54 fF ±7.8 SEM; n=8). With removal of P188, capacitance decreased over the subsequent 40 s to approximately 15 fF above baseline.

We next determined the extent to which the P188-induced capacitance increase was due to interactions of the polyethylene oxide blocks with the surface of the cell membrane, rather than insertion into the membrane. We measured whole cell capacitance changes during perfusion of a polyethylene glycol (PEG) of similar molecular weight (8400). PEG (100 µM) perfusion transiently increased whole cell capacitance above baseline values (32.8 fF ±8.7 SE), but significantly less than P188. In contrast to P188, removal of PEG from the perfusate caused the capacitance to decrease to baseline.

7. Poloxamer 188 restores disruption of membrane integrity
To assess whether P188 directly restores membrane integrity, we performed time-lapse microfluorimetry of calcein-loaded neurons before, during, and after electroporation. A single brief train (1 s duration, 5 Hz) of current pulses (0.5 A) caused rapid and complete loss of intracellular fluorescence across all cells within a 0.04 mm² area over 30–60 s (n=10 trials, 25–50 cells per trial). In contrast, perfusion of P188 (30 µM) onto neurons within 20 s after electroporation reliably arrested dye loss. Application of P188 more than 20 s after shocks did not reliably decrease the rate of dye loss.

8. Poloxamer 188 blocks plasma membrane peroxidation
To assess whether P188 reduces reactive oxygen species-induced lipid peroxidation in the plasma membrane, we assessed the effect of P188 on the rate of lipid peroxidation in single living hippocampal neurons. We induced lipid peroxidation by perfusing C11-BODIPY^581/591-loaded neurons with Fe(NH₄)₂(SO₄)₂ (200 µM) and H₂O₂ (1 mM), and monitored lipid peroxidation-dependent increases in oxidized C11-BODIPY^581/591 fluorescence over time. Perfusing neurons with HEPES-buffered saline in the absence of Fe⁴⁺ and H₂O₂ produced stable, oxidized C11-BODIPY^581/591fluorescence. Addition of Fe⁴⁺ and H₂O₂ to neurons was followed by a steady increase in fluorescence that continued until Fe⁴⁺ and H₂O₂ were removed. However, addition of P188 (30 µM) to the perfusate during Fe⁴⁺ and H₂O₂ administration abruptly and consistently decreased the rate of fluorescence increase (1.49 ± 0.48%/min Fe⁴⁺ plus H₂O₂ alone vs. 0.64 ± 0.14%/min P188 plus Fe⁴⁺ plus H₂O₂, n=16 neurons in 4 trials; P<.001).

CONCLUSIONS AND SIGNIFICANCE

This report is the first to demonstrate that an amphiphilic, tri-block copolymer provides robust neuroprotection in vitro after intense excitotoxicity or oxidative stress. The importance of neuronal necrosis after intense excitotoxicity and oxidative stress demonstrates the potential of Poloxamer 188 (and of amphiphilic, tri-block copolymer surfactants in general) in protecting neurons from important mechanisms of brain injury. The use of these molecules, therefore, represents a novel therapy for brain injuries in which these mechanisms play a central role.

Our observation of P188 insertion into the plasma membrane, as demonstrated by increases in cell surface area, is consistent with theoretical predictions of its behavior from its amphiphilic, tri-block structure. The insertion of P188 into the membrane provides a mechanism for the P188-induced restoration of membrane integrity we observed after electroporation (Fig. 2 ) and may occur via direct sealing of electropores. Similar sealing effects have been reported after application of tri-block copolymers to electroporated muscle cells and after Joule heating-induced loss of membrane integrity.

View larger version (36K): [in this window] [in a new window]   Figure 2. Schematic diagram of membrane-targeted mechanisms causing NMDA-induced neuronal death and the actions of P188 on the plasma membrane. Two P188 molecules are shown inserted into the membrane during NMDA receptor activation. One molecule is depicted physically sealing a breach of the plasma membrane, as occurs after electroporation. The other molecule is shown scavenging NMDA-induced production of reactive oxygen species and directly blocking lipid peroxidation.

Loss of membrane integrity also occurs after severe excitotoxic or oxidative stimulation through multiple mechanisms, including swelling and membrane peroxidation. P188-induced membrane sealing may contribute to the reduction of neuronal death we observed by preventing or restoring early loss of membrane integrity in susceptible neurons (Fig. 2) .

In addition to its direct effects on membrane integrity, P188 almost completely blocked lipid peroxidation induced by Fe⁴⁺ and H₂O₂. Marked neuroprotection after NMDA has been observed with malonic acid derivatives of buckminsterfullerenes, which avidly trap free radicals, and the increased neuroprotection seen with more amphiphilic derivatives has been ascribed to the greater penetration of the carbon sphere into the membrane. The hydrophobic polypropylene block of P188 may therefore mediate the profound reduction in lipid peroxidation we observed, and may stem from deep insertion into the lipid bilayer.

Many current approaches to neuroprotection, including antagonism of ligand- and voltage-gated ionic mechanisms of neuronal injury, have proved largely ineffective in clinical settings. Because the mechanisms of action are specifically directed at the plasma membrane, use of amphiphilic, tri-block copolymers may provide an alternate therapeutic approach to neuronal necrosis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0547fje ; to cite this article, use FASEB J. (February 20, 2001) 10.1096/fj.00-0547fje

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