Microscopy Research and Technique

Effects of propentofylline on CNS remyelination in the rat brainstem

Authors

  • Eduardo Fernandes Bondan,

    Corresponding author
    1. Department of Environmental and Experimental Pathology, University Paulista, São Paulo, SP, Brazil
    2. Department of Veterinary Medicine, University Cruzeiro do Sul, São Paulo, SP, Brazil
    • Correspondence to: Dr. Eduardo Fernandes Bondan, University Paulista, Rua Caconde, 125/51-01425-011 São Paulo, SP, Brazil. E-mail: bondan@uol.com.br

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  • Maria de Fátima Monteiro Martins,

    1. Department of Environmental and Experimental Pathology, University Paulista, São Paulo, SP, Brazil
    2. Department of Veterinary Medicine, University Cruzeiro do Sul, São Paulo, SP, Brazil
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  • Deborah Eileen Menezes Baliellas,

    1. Department of Veterinary Medicine, University Cruzeiro do Sul, São Paulo, SP, Brazil
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  • Caio Fernando Monteiro Gimenez,

    1. Department of Veterinary Medicine, University Cruzeiro do Sul, São Paulo, SP, Brazil
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  • Sandra Castro Poppe,

    1. Department of Environmental and Experimental Pathology, University Paulista, São Paulo, SP, Brazil
    2. Department of Veterinary Medicine, University Cruzeiro do Sul, São Paulo, SP, Brazil
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  • Maria Martha Bernardi

    1. Department of Environmental and Experimental Pathology, University Paulista, São Paulo, SP, Brazil
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  • REVIEW EDITOR: Dr. Peter Saggau

ABSTRACT

Propentofylline (PPF) is a xanthine derivative with pharmacological effects distinct from those of the classical methylxanthines. It depresses activation of microglial cells and astrocytes which is associated with neuronal damage during neural inflammation and hypoxia. The aim of this study was to evaluate whether PPF had the capacity of affecting glial cells behavior during the process of demyelination and remyelination following ethidium bromide (EB) gliotoxic injury. EB injection into the CNS is commonly used as an experimental demyelinating model inducing local oligodendroglial and astrocytic death, which results in primary demyelination, blood–brain barrier and glia limitans disruption and Schwann cells invasion. Sixty Wistar rats were divided into four different groups receiving 10 microlitres of 0.1% EB or 0.9% saline solution into the cisterna pontis and treated or not with the xanthine. PPF treatment was done using 12.5 mg/kg/day by the intraperitonial route for 31 days of the experimental period. The rats were euthanized from 7 to 31 days after EB injection and brainstem sections were collected and processed for light and transmission electron microscopy studies. Results from both groups were compared by using a semi-quantitative method developed for documenting in semithin sections the extent and nature of remyelination of demyelinating lesions. Results showed that PPF administration after EB injection significantly increased both oligodendroglial and Schwann cell remyelination at 31 days (mean remyelination scores of 3.67 ± 0.5 for oligodendrocytes and 1.27 ± 0.49 for Schwann cells) compared to untreated animals (scores of 3.19 ± 0.57 and 0.90 ± 0.33, respectively). Microsc. Res. Tech. 77:23–30, 2014. © 2013 Wiley Periodicals, Inc.

INTRODUCTION

Propentofylline [PPF, 3-methyl-1-(5′-oxohexyl)−7-propylxanthine] is a xanthine derivative with pharmacological effects distinct from those of the classical methylxanthines theophylline and caffeine (Koriyama et al., 2003). In vitro and in vivo studies have demonstrated profound neuroprotective, antiproliferative, and anti-inflammatory effects of PPF in several experimental models in animals (Sweitzer and DeLeo, 2011). Clinically, it has shown efficacy in degenerative vascular dementia (Kittner et al., 1997) and as a potential adjuvant treatment to Alzheimer's disease (Bachynsky et al., 2000; Wilkinson, 2001), schizophrenia (Salimi et al., 2008), and multiple sclerosis (Suzumura et al., 2000). It probably depresses activation of microglial cells and astrocytes which is associated with neuronal damage during inflammation and hypoxia and consequently decreases glial production and release of damaging proinflammatory factors (Sweitzer and DeLeo, 2011). In such context, the aim of this study was to evaluate whether PPF had also the capacity of affecting glial cell behavior during the process of demyelination and remyelination following gliotoxic injury induced by ethidium bromide (EB). It is known that focal EB injection into the central nervous system (CNS) is a well-established experimental demyelinating model inducing local oligodendroglial and astrocytic death and resulting in primary demyelination, blood–brain barrier and glia limitans disruption, as well as Schwann cell invasion from peripheral nerves (Blakemore, 1982; Bondan et al., 2000, 2006, 2011; Bondan and Martins, 2013; Graça and Blakemore, 1986; Pereira et al., 1998; Yajima and Suzuki, 1979).

METHOD

This experiment was approved by the Ethics Commission of the University Paulista (protocol number 023/11). Sixty male Wistar rats, 4- to 6-month old, were divided into the following groups: I—rats injected with 10 µL of 0.1% EB solution into the cisterna pontis and treated with PPF (n = 20); II—rats injected with EB and not treated with PPF (n = 20); III—rats injected with 10 µL of 0.9% saline solution and treated with PPF (n = 10); and IV—rats injected with saline solution and not treated with PPF (n = 10).

The rats were anaesthetized with ketamine and xylazine (5:1, 0.1 mL/100 g) and a burr hole was made on the right side of the skull, 8 mm rostral to the fronto-parietal suture. Injections were performed freehand using a Hamilton syringe, fitted with a 35° angled polished gauge needle into the cisterna pontis, an enlarged subarachnoid space below the ventral surface of the pons.

The animals were kept under controlled light conditions (12 h light–dark cycle) and water and food were given ad libitum during the experimental period.

Rats from groups I and III were daily treated with PPF solution (20 mg/mL, Agener União Química, São Paulo, SP) by the intraperitoneal route using 12.5 mg/kg/day during the experimental period.

For groups I and II, three animals were anaesthetized and submitted to intracardiac perfusion with 4% glutaraldehyde in 0.1 M Sorensen phosphate buffer (pH 7.4) at 7, 11, 15, and 21 days post-injection (p.i.) and eight were perfused at 31 days p.i. For groups III and IV, two animals were submitted to perfusion at each period previously mentioned. Thin slices of the brainstem (pons and mesencephalon) were collected and post-fixed in 1% osmium tetroxide, dehydrated with graded acetones, and embedded in Araldite 502 resin, following transitional stages in acetone. Thick sections were stained with 0.25% alkaline toluidine blue. Selected areas were trimmed and thin sections were stained with 2% uranyl acetate and lead citrate and examined using a Philips EM-201 transmission electron microscope.

Comparison between the final balance of myelin repair in PPF treated and untreated rats was assessed using the semi-quantitative method developed by Gilson and Blakemore (1993). Three semithin sections from each animal at 31 days after EB injection from groups I and II were examined for the presence of axons remyelinated by oligodendrocytes and Schwann cells, as well as for demyelinated axons. Remyelination by either Schwann cells or oligodendrocytes was identified using morphological criteria previously described (Blakemore, 1982; Bondan et al., 2000, 2006; Graça and Blakemore, 1986; Pereira et al., 1998; Yajima and Suzuki, 1979). The proportion of each was estimated in a scale ranging from 0 to 5. A lesion in which all axons were remyelinated by Schwann cells would have a Schwann cell (S) score of 5; an oligodendrocyte (O) score of 0; and a demyelination (D) score of 0. If 60% of the demyelinated axons were remyelinated by oligodendrocytes and 20% by Schwann cells with the remaining axons being demyelinated, then the lesion was assigned a score of O-3, S-1, D-1. To compare remyelination scores from rats treated or not with PPF 31 days after EB injection, a lesion repair profile of O versus S remyelination was made, providing an adequate graphical representation of each group. The mean O and S scores ±2 SEM enclose a domain which represent an average of repair. Non-overlapping domains in these graphic representations indicate significantly distinct results.

RESULTS

EB-induced Lesions in Rats From Groups I and II

By 7–11 days p.i., the examination of semithin and ultrathin sections from both groups revealed the appearance of demyelinating lesions in the ventral surface of the pons and mesencephalon. Two areas with very distinct morphological characteristics were noticed. The central region presented an expanded extracellular space, with demyelinated axons (Fig. 1A), foamy macrophages (Fig. 1B), myelin-derived membranes, and some lymphocytes. No astrocytic processes were found in this site. At peripheral locations, phagocytic cells were less conspicuous and by day 11 p.i. some cells with morphological resemblance to oligodendrocytes were seen over the edges of the lesions, but without any indication of remyelinating activity. Thinly remyelinated axons could be seen at day 15 p.i., some associated with oligodendrocytes (Fig. 2), others related to Schwann cells. Schwann cells were associated with one or multiple demyelinated axons or already forming thin myelin lamellae around single axons in astrocyte-free areas (Fig. 3). On the other hand, oligodendrocytes began to form slight myelin sheaths in areas partially filled with astrocytic processes. In association with peripheral astrogliosis (Fig. 4), pial cell infiltration was noted from 15 to 31 days after EB injection and, although rare, some axons with signs of degeneration (Fig. 5) persisted until day 31 p.i. Differences between rats treated or not with PPF clearly appeared from day 21 p.i. Rats treated with PPF presented a greater proportion of oligodendrocyte remyelinated axons (Figs. 6A and 6C) when compared to the untreated ones (Figs. 6B and 6D). Schwann cell remyelination (Figs. 7A and 7B) was also easily found at subpial and perivascular locations.

Figure 1.

A, B: Electronmicrographs of a central area containing demyelinated axons (d) in (A) and macrophages (m) in intense phagocytic activity in (B) at 11 days after EB injection. Note in (A) a tight junction (arrowhead) in a blood vessel. Group II. (A) Bar = 1 µm; (B) Bar = 4 µm.

Figure 2.

Electronmicrograph showing axons in initial stage of remyelination by oligodendrocytes (O) at 15 days after EB injection. Group II. Bar = 1 µm.

Figure 3.

Electronmicrograph presenting Schwann cells (S) associated to demyelinated axons or already in remyelination at 15 days after EB injection and near to macrophages (m) and blood vessels (v). Note the portions of Schwann cell cytoplasm covering these axons. Group II. Bar = 3 µm.

Figure 4.

Electronmicrograph from a peripheral site showing demyelinated axons (d) around a blood vessel (v) and a hypertrophic astrocyte process (a) at 21 days post-lesion. Group II. Bar = 1 µm.

Figure 5.

Electronmicrograph of degenerating axons (a) next to demyelinated axons (d) and macrophages (m) at 21 days after EB injection. Group II. Bar = 2 µm.

Figure 6.

A, B: Photomicrographs from semithin sections belonging to groups I (PPF-treated group) and II (untreated group) 21 days after EB injection. (A) Group I. Bar = 25 µm; (B) Group II. Bar = 25 µm. Note, in (A), an area around a blood vessel (v) in which the majority of axons are already remyelinated (r), while, in (B), there are still many demyelinated axons (d) near macrophages (m) and pial cell nests (p). Very few axons are in initial stages of remyelination (arrow). C, D: Electronmicrographs comparing two peripheral areas at 21 days post-lesion. Observe the thin and newly formed myelin sheaths (arrowhead) around oligodendrocyte remyelinated axons (r) in (C) (group I; Bar = 1 µm) and the many still demyelinated axons (d) in (D) (group II; Bar = 1 µm).

Figure 7.

A, B: Electronmicrographs presenting axons remyelinated by an oligodendrocyte (O) and by a Schwann cell (S) in astrocyte-free areas at 31 days post-lesion. (A) Group I. Bar = 5 µm; (B) Group I. Bar = 2 µm.

Saline-induced Lesions in Rats From Groups III and IV

By 7 days p.i., discrete lesions restricted to the pons and along the needle track were noted in semithin sections, due to the surgical procedure. Ultrastructural examination of the lesions showed a light and focal expansion of the extracellular space containing phagocytic cells, some loose lamellae and few myelin debris. A small number of fibers showed periaxonal edema and morphological aspects of degeneration, but there was no sign of primary demyelination or loss of glial cells.

Score Comparison Between Groups I and II at 31 Days

The semi-quantitative method developed by Gilson and Blakemore (1993) was used in order to confirm the apparent difference in remyelination (noted by both light and transmission electron microscopy) between EB-injected rats from groups I and II. Remyelination scores at 31 days are represented in Table 1 and the respective diagrams in Figure 8.

Table 1. Remyelination scores 31 days after ethidium bromide (EB) injection in groups I (treated with PPF) and II (not treated with PPF) according to the semi-quantitative method developed by Gilson and Blakemore (1993) for semithin sections
 Group I (with PPF)Group II (without PPF)
Sections per animalOSDOSD
  1. See Material and Methods for explanation of scoring. O, axons remyelinated by oligodendrocytes; S, axons remyelinated by Schwann cells; D, demyelinated axons; SD, standard-deviation; SEM, standard error of the mean.

13.51.50.03.50.51.0
12.52.00.53.01.50.5
13.51.50.03.51.50.0
23.51.50.03.01.01.0
23.02.00.02.01.51.5
24.01.00.03.51.00.5
33.51.00.52.51.01.5
33.51.50.04.00.50.5
34.00.50.53.50.51.0
44.50.50.03.01.01.0
44.01.00.02.01.02.0
43.51.50.04.00.50.5
54.01.00.03.51.00.5
54.01.00.03.01.01.0
53.51.50.03.50.51.0
64.01.00.03.01.01.0
63.02.00.03.51.00.5
63.51.50.02.51.01.5
74.50.50.04.00.50.5
73.51.50.03.50.51.0
74.50.50.03.01.01.0
84.01.00.03.51.00.5
83.51.50.03.50.51.0
83.02.00.02.51.01.5
MEAN3.671.270.063.190.900.92
SD0.50.490.170.570.330.46
SEM0.110.100.040.120.070.10
Figure 8.

Diagrams of oligodendrocyte (O) versus Schwann cell (S) remyelination scores in rats treated with PPF (group I) or not (group II) 31 days post-lesioning. The areas within circles represent mean ± 2 SEM and are considered as domains that represent an average of repair. Non-overlapping repair domains indicate significantly different results. The majority of axons are remyelinated by oligodendrocytes. n = number of observations.

PPF-treated rats presented significantly increased scores (means of 3.67 ± 0.5 for oligodendrocytes and 1.27 ± 0.49 for Schwann cells) while the untreated rats had mean remyelination scores of 3.19 ± 0.57 for oligodendrocytes and 0.90 ± 0.33 for Schwann cells.

Non-overlapping domains (defined as areas within circles graphically representing mean ± 2 SEM) in the repair profiles from groups I and II indicated that the results were significantly different.

DISCUSSION

Known mechanisms of PPF include inhibition of cyclic AMP (cAMP) and cyclic GMP phosphodiesterases (PDE) and action as a reuptake inhibitor for the purine nucleoside and neurotransmitter adenosine by blocking the action of membrane nucleoside transporters (ENTs). This may lead to increased intracellular cAMP levels and greater extracellular concentrations of adenosine (Sweitzer and DeLeo, 2011), which stimulates adenosinergic neurotransmission and adenosine 2 (A2) receptor-mediated cAMP synthesis (Schubert et al., 2000).

In the CNS, PPF may serve as a glial modulator, with direct actions on microglia, and dose dependently decreases microglial proliferation and expression of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), as seen in response to lipopolysaccharide (LPS) stimulation in vitro (Si et al. 1996, 1998; Yoshikawa et al., 1999).

In experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS), inflammatory infiltrates mainly consist of T lymphocytes and macrophages which are regarded as the most important effector cells. During the induction and effector phase of such diseases these cells secrete pro-inflammatory cytokines and cytotoxic factors characteristic of an immune response of the type 1 cytokine pattern such as TNF-α and β, interleukin-2 (IL-2) and interferon-δ (IFN-δ) (Hartung, 1995).

In the EB experimental model, lymphocytes are commonly found within demyelinating lesions, sometimes contacting myelin debris in the extracellular space as well as activated macrophages containing phagocytosed myelin, in a relationship suggestive of antigenic recognition (Bondan et al., 2000; Graça and Blakemore, 1986). Their presence was credited to the general inflammatory response induced by the gliotoxin in the nervous tissue and their income probably facilitated by the early disappearance of perivascular astrocytes after EB injection in the lesion site (Bondan et al., 2000).

The lack of evidence of an immune-mediated response to explain the lymphocytic participation in the EB model cannot be taken as unequivocal evidence of its non-occurrence, as there was blood–brain barrier disruption and consequent loss of the immunologic privilege site condition. It can not be ruled out the possibility that the entry of lymphocytes in the course of CNS inflammation response can generate a local and limited oligoclonal response, as a result of the exposition of CNS components to those infiltrating immune cells (Leibowitz and Hughes, 1983).

Regulation of the production of TNF-α and other cytokines by leukocytes includes the adenylate cyclase-cAMP-protein kinase pathway, which also affects the activity of a great number of other cell types (Kammer, 1988).

Intracellular levels of the second messenger cAMP can be elevated by activation of the adenylate cyclase or by inhibition of cAMP-degrading PDE, a group of enzymes with at least five isoenzyme families. Elevation of cAMP in leukocytes mainly down-regulates immune responses. Thus, inhibition of TNF-α synthesis or secretion of the T helper cell type 1 (Th1)-derived cytokines IL-2, IL-12, and IFN-δ has been described for a number of PDE inhibitors (PDEIs), including PPF, and for activators of adenylate cyclase, such as prostaglandins and prostacyclin analogues (Jung et al., 1997).

As previously observed, the inhibition of IL-2 secretion induced by cyclosporine (CsA) stimulated oligodendrocyte remyelination in rats (Bondan et al., 2011), even in diabetic rats (Bondan and Martins, 2013). On the other hand, Saneto et al. (1986) noticed that IL-2 presence was capable of inhibiting the proliferation of oligodendrocyte progenitor cells (OPCs) in vitro even though stimulating their maturation in adult oligodendrocytes. Thus, the lack of IL-2 in the CNS inflammatory environment could allow the migration and division of OPCs, explaining the increased remyelination with CsA (Bondan et al., 2011).

Yoshiwawa et al. (1999) reported that PPF, a type III–IV specific PDEI, although decreasing in a dose-dependent manner the production of the inflammatory cytokines TNF-α, IL-1, and IL-6 by mouse microglia stimulated by LPS in vitro, increased up to two or three times the production of IL-10, an inhibitory cytokine. Down-regulated expression of mRNA encoding the LPS-induced cytokines TNF-α, IL-1, and IL-6 was confirmed at an mRNA level by RT-PCR, as well as IL-10 mRNA up-regulation. It is known that IL-1 activates T lymphocytes during antigen presentation, induces adhesion molecules in endothelial cells, promotes astrocyte release of TNF-α and IL-6, and stimulates nitric oxide synthase (NOS) production in these cells. Activity of IL-1 is relatively high in MS plaques in the acute phase, suggesting an important role in CNS demyelination. On the contrary, IL-10 is recognized as an inhibitory cytokine, which hinders the cytokine production of Th1 lymphocytes and also inhibits the activation of macrophages and microglia induced by IFN-δ (Yoshikawa et al., 1999). IL-10 has also been shown to suppress EAE (Johns et al., 1991), although Jung et al. (1997) did not observe that preventive treatment of Lewis rats with PPF have significantly ameliorated clinical signs of EAE actively induced by immunization with myelin basic protein (MBP) in complete Freund's adjuvant, even with moderate inhibition of TNF-α by macrophages limited to a few hours after the injection.

The concept of a “bystander demyelination” was based on the demonstration that oligodendrocytes in tissue culture were particularly sensitive to factors such as reactive oxygen species (ROS) (Griot et al., 1989), TNF (Selmaj and Raine, 1988), and complement (Scolding et al., 1990). It was accepted that inflammation would create a milieu with potentially damaging molecules to myelin forming cells—not only mature cells would be destroyed, but recruited cells (which differentiate to replace the lost cells) would also be vulnerable. Thus it is possible that macrophage and lymphocyte products during the inflammatory response triggered by the EB injection may provide a greater harmful influence to the nervous tissue than the early gliotoxin injection. Therefore anti-inflammatory effects performed by PPF may be predominantly supportive to remyelination.

It has been hypothesized that pathogenic mechanisms involved in many CNS pathological conditions, including demyelination, probably involve a Ca++-dependent and excessive activation of glial cells in a way that the reinforcement of endogenous homeostatic regulators may provide a neuroprotective therapy which can possibly handle with the complexity of such processes (Schubert et al., 1997a, 1997b).

Adenosine is an ancient molecular signal that acts on both neurons and glial cells. In the latter, the prevalent effect of adenosine is its regulatory influence on the Ca++- and cAMP-dependent molecular signaling that determines the cellular proliferation rate, the differentiation state, and related functions (Schubert et al., 1997a). A strengthening of the cAMP signaling, which can be achieved by adenosine agonists and by PPF, stimulated the mRNA production of neurotrophic factors in astrocytes, apparently preventing a deleterious and secondary astrocytic activation caused by previous microglial up-regulation (Schubert et al., 2000).

In cultured microglial cells, several days' treatment with adenosine agonists or PPF increased apoptosis in activated microglial cells and strongly inhibited their proliferation rate, their spontaneously occurring transformation into macrophages and, particularly, the high formation of ROS (Schubert et al., 1997a, 1997b).

In addition, decreased activation of astrocytes and microglia, as shown by reduced GFAP and OX-42 expression, respectively, was observed in vivo after spinal cord injury in rats treated with PPF (Young et al., 2008).

An important clinical feature of PPF is its minimal adverse effect profile, demonstrated in multiple clinical trials (Jacobs et al., 2011, 2012).

In our study, there was no apparent difference regarding astrocyte, lymphocyte, or macrophage presence in the EB-induced lesions between both groups (treated or not with PPF). Lesions were similar to those found in this toxic model (Blakemore, 1982; Bondan et al., 2000; Graça and Blakemore, 1986; Pereira et al., 1998; Yajima and Suzuki, 1979); however rats treated with PPF presented lesions that resembled those observed with CsA treatment (Bondan et al., 2011; Bondan and Martins, 2013), with a higher density of oligodendrocytes over the edges of the lesions and increased remyelination. The precise mechanisms by which these beneficial PPF effects occur remain obscure. Although it has been accepted that drugs which elevate extracellular adenosine and/or block the degradation of cyclic nucleotides, like PPF, may be used to counteract glia-related damage in CNS pathological processes (Schubert et al., 2000), further investigations must be conducted, especially concerning demyelinating conditions.

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