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. 2017 Jun 14;118(4):2059–2069. doi: 10.1152/jn.00680.2016

Prevention and reversal of latent sensitization of dorsal horn neurons by glial blockers in a model of low back pain in male rats

Juanjuan Zhang 1,2, Siegfried Mense 1, Rolf-Detlef Treede 1, Ulrich Hoheisel 1,✉
PMCID: PMC5626892  PMID: 28615336

Activated microglia and astrocytes mediate the latent sensitization induced by nerve growth factor in dorsal horn neurons that receive input from deep tissues of the low back. These processes may contribute to nonspecific low back pain.

Keywords: nonspecific low back pain, nerve growth factor, electrophysiology, glial cell activation, latent sensitization

Abstract

In an animal model of nonspecific low back pain, recordings from dorsal horn neurons were made to investigate the influence of glial cells in the central sensitization process. To induce a latent sensitization of the neurons, nerve growth factor (NGF) was injected into the multifidus muscle; the manifest sensitization to a second NGF injection 5 days later was used as a read-out. The sensitization manifested in increased resting activity and in an increased proportion of neurons responding to stimulation of deep somatic tissues. To block microglial activation, minocycline was continuously administered intrathecally starting 1 day before or 2 days after the first NGF injection. The glia inhibitor fluorocitrate that also blocks astrocyte activation was administrated 2 days after the first injection. Minocycline applied before the first NGF injection reduced the manifest sensitization after the second NGF injection to control values. The proportion of neurons responsive to stimulation of deep tissues was reduced from 50% to 17.7% (P < 0.01). No significant changes occurred when minocycline was applied after the first injection. In contrast, fluorocitrate administrated after the first NGF injection reduced significantly the proportion of neurons with deep input (15.8%, P < 0.01). A block of glia activation had no significant effect on the increased resting activity. The data suggest that blocking microglial activation prevented the NGF-induced latent spinal sensitization, whereas blocking astrocyte activation reversed it. The induction of spinal neuronal sensitization in this pain model appears to depend on microglia activation, whereas its maintenance is regulated by activated astrocytes.

NEW & NOTEWORTHY Activated microglia and astrocytes mediate the latent sensitization induced by nerve growth factor in dorsal horn neurons that receive input from deep tissues of the low back. These processes may contribute to nonspecific low back pain.

little is known about the contribution of glial cells to the development of low back pain. A better understanding of glial mechanisms in this context is important because chronic low back pain is one of the most common types of clinically relevant pain (Balagué et al. 2012) and glial cells are demonstrated to be involved in other types of chronic pain such as neuropathic and inflammatory pain (Malcangio 2016).

Often the cause of low back pain cannot be determined by conventional clinical diagnostic methods (nonspecific low back pain). Low back muscles and fasciae are potential sources of nonspecific low back pain (Stecco et al. 2011), and experimental evidence for such a role has been published from studies on animals (Hoheisel et al. 2011, 2013; Taguchi et al. 2008) and humans (Deising et al. 2012; Gibson et al. 2009; Schilder et al. 2014, 2016).

We recently demonstrated that dorsal horn neurons processing nociceptive input from the low back can acquire a state of latent sensitization after a single injection of nerve growth factor (NGF) into a low back muscle (Hoheisel et al. 2013). The NGF injection sensitizes dorsal horn neurons transiently (maximally 3 days). After this period, the neurons are in a state of latent sensitization; the term means that the response behavior of the dorsal horn neurons is completely normal, but they are easier to sensitize by a subsequent NGF injection in the muscle. When a second NGF injection is given in that state of latent sensitization, a stronger sensitization of the neurons ensues (Hoheisel et al. 2013). This behavior of dorsal horn neurons resembles the recently described priming of peripheral nociceptors by repeated injections of prostaglandins or acidic solutions into limb muscles or intraplantar injections of inflammatory mediators (DeSantana and Sluka 2008; Ferrari et al. 2010; Hendrich et al. 2013; Reichling and Levine 2009).

There is substantial evidence that glia activation leads to altered neuronal excitability (Milligan and Watkins 2009; Xanthos and Sandkühler 2014). Substances released by activated glial cells (e.g., brain-derived neurotrophic factor and TNF-α) cause a hyperexcitability of spinal neurons and hyperalgesia in behavioral experiments (Clark et al. 2015; Coull et al. 2005; McMahon and Malcangio 2009; Milligan and Watkins 2009). Activation of spinal glia was found in animal models of neuropathic pain (Ledeboer et al. 2005; Milligan et al. 2003; Tsuda et al. 2013) and inflammatory pain (Chacur et al. 2009; Raghavendra et al. 2003).

In the present study, the role of glial cells in the sensitization of dorsal horn neurons processing input from soft low back tissues was investigated using extracellular recordings from single neurons. The influence of microglia on the sensitization process was tested with continuous intrathecal administration of minocycline (Chacur et al. 2009; Clark et al. 2015; Ledeboer et al. 2005; Raghavendra et al. 2004), which specifically inhibits microglia and has no effects on astrocytes or neurons (Tikka and Koistinaho 2001). The unspecific glial cell inhibitor fluorocitrate was administrated that also blocks astrocyte activation (Clark et al. 2007; Gerhold et al. 2015). To sensitize dorsal horn neurons, repeated injections of NGF into the multifidus muscle were given (Hoheisel et al. 2013). Histological observation proved that no signs of inflammation were found in the injected muscle (Hoheisel et al. 2013). Therefore, such NGF injections are a noninflammatory animal model to study mechanisms that may be involved in nonspecific low back pain in patients. The release of NGF is closely connected to many painful muscle disorders and to nonpainful but functionally overloaded muscles. In our experimental animals, the two NGF injections did not elicit pain-related behavior but caused a long-lasting hyperalgesia in the injected low back muscle without producing any visible structural changes (Hoheisel et al. 2013).

In this study, the hypothesis was tested that spinal glial cells have to be blocked to prevent or reverse the latent sensitization of dorsal horn neurons.

MATERIALS AND METHODS

Animals

All experiments were performed on 28 adult male Sprague-Dawley rats (body weight 300–400 g). Sex differences are not the topic of the present study, but they are known to exist (Mogil et al. 2010; Sorge et al. 2015; Taves et al. 2016). The experimental procedure and the number of animals used were approved by the regional board Karlsruhe and carried out in accordance with the German law on the protection of animals and with the ethical proposals of the International Association for the Study of Pain (Zimmermann 1983).

There were three dropouts during the study: in one animal the intrathecal catheter was clogged by a tissue clot, and in a second animal the inserted catheter had injured dorsal roots (see Implantation of the Chronic Intrathecal Catheter); in a third animal the blood pressure was too low to record central neuronal activity (<80 mmHg, see Recording of Spinal Dorsal Horn Neurons). These three animals were excluded from the data evaluation and replaced.

Injection of NGF into the Multifidus Muscle

Injections of 50 µl NGF solution (human recombinant; Calbiochem Merck, Darmstadt, Germany; Hoheisel et al. 2011; 2013) at a concentration of 0.8 µM were made into the multifidus muscle 3 mm lateral of the spinous process L5 (Fig. 1C, for details see Hoheisel et al. 2013). NGF was dissolved in PBS, and the solution was adjusted to pH 7.2–7.3. Two injections of NGF were given at an interval of 5 days (Fig. 1, A and B) and were made at the same site. Directly after the second NGF injection, the recording of dorsal horn neurons started (Fig. 1). Injections of 50 µl PBS solution served as a negative control (only PBS group, see treatment groups). The NGF concentration used is known to cause hyperalgesia when injected intramuscularly in animals or humans (Hoheisel et al. 2013; Svensson et al. 2003; Weinkauf et al. 2015).

Fig. 1.

Fig. 1.

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Intrathecal administration. A: schedule of intrathecal administration starting 1 day before the first nerve growth factor (NGF) or PBS injection. B: intrathecal administration starting 2 days after the first NGF injection. Open bars indicate intrathecal administration via the osmotic pump. The duration of intrathecal treatment differs between prevention [mino-pre group (A)] and reversal protocols [mino-post group and fluoro-post group (B)]. In A the intrathecal administration started before the first NGF injection that induced the neuronal latent sensitization, whereas in B the blockers were given during the state of latent sensitization, which was caused by the first NGF injection. Upward arrows indicate time of NGF or PBS injection. Solid bars represent recording period of 4 h, starting immediately after the second NGF or PBS injection. C: location of the lumbar puncture (needle) between the vertebrae L5 and L6. The catheter was inserted through the needle (arrow) on the right of the spinous processes and pushed craniad. The site of NGF or PBS injection into the multifidus muscle (marked in black) was located on the left side of spinous process L5. Recordings were made at the level of vertebra T12 in spinal segment L2.

Implantation of the Chronic Intrathecal Catheter

Under sterile conditions, an intrathecal catheter was implanted in the lumbar spinal canal (Chacur et al. 2009). The animals were deeply anaesthetized with a mixture of ketamine and xylazine (100 mg/kg and 7.5 mg/kg ip; Bela-Pharm, Vechta, Germany and Bayer Vital, Leverkusen, Germany, respectively). After a small skin incision, a sterile 18-gauge needle was inserted between the lumbar vertebrae L5 and L6 on the right side of the spinous processes (see Fig. 1C). A sterile polyethylene catheter (PE-10 tube, 0.28-mm ID/0.61-mm OD) was inserted through the needle and carefully pushed craniad up to the level of the lumbosacral enlargement. To ensure that there was a sufficient intrathecal concentration of the test solution immediately after catheter implantation, an initial bolus of 12 µl test solution was delivered (Chacur et al. 2009). After this initial bolus, the catheter was connected to an osmotic pump (model 2002; Alzet, Cupertino, CA) filled with the test solution (see Treatment Groups). The pumps used had an infusion rate of 0.5 µl/h, resulting in a daily intrathecal fluid administration of 12 µl. The correct intrathecal catheter placement was verified when the laminectomy to expose the spinal segments was performed (see Recording of Spinal Dorsal Horn Neurons).

Intrathecally Applied Blocker of Glia Cell Activation

Minocycline.

Minocycline hydrochloride was obtained from Sigma-Aldrich (Hamburg, Germany). After an initial bolus of 200 µg in 12 µl fluid directly after catheter implantation, 200 µg minocycline per day was administered continuously with the osmotic pump (Fig. 1, A and B; mino-pre and mino-post groups). Minocycline is widely regarded to be a specific blocker of microglia (Tikka and Koistinaho 2001); however, nonspecific side effects of minocycline were also discussed recently (Möller et al. 2016).

Fluorocitrate.

DL-fluorocitrate acid barium salt was obtained from Sigma-Aldrich. After an initial bolus of 0.83 µg in 12 µl fluid, fluorocitrate was administrated continuously with the osmotic pump (Fig. 1B, fluoro-post group). Fluorocitrate was used as a rather unspecific blocker of glial cells including microglia and astrocytes (Clark and Malcangio 2014; Gruber-Schoffnegger et al. 2013).

Both blockers of glial activation were diluted in sterile artificial cerebrospinal fluid (Philippu, 1984), and the solution was adjusted to pH 7.0. The concentrations used were adopted from the literature and adjusted to values that are known to block glial cell activation effectively in comparable experimental approaches (minocycline: Chacur et al. 2009; Ledeboer et al. 2005; Tikka and Koistinaho 2001; fluorocitrate: Cao et al. 2014; Gruber-Schoffnegger et al. 2013; Sung et al. 2012). Intrathecally applied artificial cerebrospinal fluid served as a control (PBS group and NGF group, Fig. 1A).

Treatment Groups

Twenty five animals were divided into five treatment groups with 5 animals each. In two groups artificial cerebrospinal fluid was applied intrathecally.

PBS group.

The animals received two PBS injections into the multifidus muscle, and artificial cerebrospinal fluid was administered intrathecally for 6 days starting 1 day before the first PBS injection was given (Fig. 1A).

NGF group.

Two intramuscular NGF injections were given into the multifidus muscle, and artificial cerebrospinal fluid was applied intrathecally for 6 days starting 1 day before the first NGF injection (Fig. 1A).

In three further groups the blockers of glia cell activation were administered intrathecally.

Mino-pre group.

Two NGF injections were given into the multifidus muscle, while minocycline was applied intrathecally for 6 days starting 1 day before the first NGF injection (Fig. 1A).

Mino-post group.

Two NGF injections were given into the multifidus muscle, but minocycline was administered intrathecally for 3 days starting 2 days after the first NGF injection (Fig. 1B). The mino-post group differed from the mino-pre group in that minocycline was administered later, after the first NGF injection.

Fluoro-post group.

Two intramuscular NGF injections were given, but, instead of minocycline, fluorocitrate was administered intrathecally for 3 days starting 2 days after the first NGF injection (Fig. 1B).

Recording of Spinal Dorsal Horn Neurons

The animals were deeply anaesthetized with thiopental sodium (Trapanal; Inresa, Freiburg im Breisgau, Germany), 100 mg/kg ip initially, followed by an intravenous infusion (external jugular vein) of 10–20 mg·kg−1·h−1 thiopental sodium using an infusion pump to maintain a deep and constant level of anesthesia (no flexor reflexes or blood pressure reactions exceeding 10 mmHg occurred to noxious stimuli). Muscular relaxation was induced with pancuronium bromide (Inresa, 0.5 mg·kg−1·h−1 iv). Mean arterial blood pressure measured in the right common carotid artery and body core temperature were continuously monitored and kept at physiological levels (above 80 mmHg, 37–38°C). The animals were artificially ventilated with a gas mixture of 47.5% O2-2.5% CO2-50% N2 (Hoheisel et al. 2013). A laminectomy was performed to expose the spinal segments L1 to L5. The laminectomy did not affect the caudal back muscles and the overlying fascia (Hoheisel et al. 2013; Taguchi et al. 2008). At the end of the final experiments, the animals were killed under deep anesthesia with an overdose of the anesthetic.

Extracellular recordings from dorsal horn neurons were made in the spinal segment L2, which receives strong input from deep low back tissues (Hoheisel et al. 2011, 2013). Recordings were made with glass microelectrodes filled with 5% NaCl (10–38 MΩ). Microelectrode penetrations were made to a depth of 1,000 µm. As a search stimulus, the dorsal roots L3 to L5 were electrically stimulated together with a single electrode (intensity 5 V, width 0.3 ms, repetition rate 0.33 Hz). All dorsal horn neurons giving stable responses to this stimulus were accepted for the study.

Identification of Receptive Fields and Neuron Classification

The receptive fields of the neurons were identified with mechanical stimulation of low back structures (muscle, fascia, skin) plus the left hind limb, hip, lateral abdominal wall, and tail. As innocuous stimuli, touch with an artist’s brush and moderate pressure (stimulus force: 1 N) with a blunt probe were used; as noxious stimuli, pinching with a sharpened watchmaker’s forceps (skin, thoracolumbar fascia) or noxious pressure (stimulus force: 10 N) with a blunt probe (muscle) were used. In some cases, as a noxious chemical stimulus, 5% NaCl (50 µl) was injected into muscles underlying a mechanosensitive cutaneous or fascial receptive field.

In the quantitative evaluation, the input of a given neuron was the criterion. When a certain input is mentioned (e.g., skin), the input could exist with or without input from another source. When a neuron responded to touching or pinching of the skin, it was considered a neuron having skin input. In neurons that responded to moderate or noxious pressure applied to deep somatic structures (e.g., muscle) but did not respond to brushing and pinching of the overlying skin, the receptive field was considered to be in the muscle or other deep structures. The deep somatic tissues of the low back could be tested directly because the skin of that area was opened. To identify a receptive field in the thoracolumbar fascia, it was pinched with a watchmaker’s forceps. Intramuscular injections of 5% NaCl were given to identify receptive fields in a muscle underneath a mechanosensitive field in the fascia or skin. Size and location of the receptive field (in case of muscular ones, their projection on the surface) were recorded on a standard outline of the rat body.

The search for receptive fields was carried out following a strict protocol in all experimental groups. The responsiveness of each neuron was tested by stimulating the body regions in a fixed order (Hoheisel and Mense 2015) starting with the toes and followed by metatarsus, heel, lower leg, knee, thigh, base of the tail, low back, and lateral abdomen (Fig. 2).

Fig. 2.

Fig. 2.

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Proportion of dorsal horn neurons with deep or cutaneous input. A: neurons with input from deep tissues (e.g., muscle, fascia). B: neurons with skin input. There were two control groups, PBS (open bar, group 1), two PBS (vehicle) injections into the multifidus muscle; NGF (solid bar, group 2), two injections of NGF into the muscle. The injections were given at 5-day intervals. In both control groups artificial cerebrospinal fluid was administered intrathecally. Test groups with intrathecal administration of minocycline or fluorocitrate included the following: mino-pre (hatched bar, group 3) (2 NGF injections and minocycline administration starting before the first NGF injection), mino-post (hatched bar, group 4) (2 NGF injections and minocycline administration after the first NGF injection), and fluoro-post (gray bar, group 5) (2 NGF injections and fluorocitrate administration after the first NGF injection). The numbers underneath the bars are those of the neurons from which the bars were constructed. The P value indicates statistically significant differences between the treatment groups (Fisher’s exact probability test). The insert illustrates the protocol of searching for receptive fields in a fixed order (Hoheisel et al. 2015). 1, toes; 2, metatarsus; 3, heels; 4, lower leg; 5, knee; 6, thigh; 7, base of tail; 8, low back; 9, lateral abdomen.

The resting (ongoing) discharge of each neuron was determined before it was tested with experimental stimuli. A neuron was defined as having resting activity if it fired ≥1 action potential per minute.

Data Analysis

Proportions of neurons were compared with Fisher’s exact probability test. Comparison between treatment groups was made using the U-test of Mann and Whitney. A probability level of <5% (two-sided) was regarded as significant.

RESULTS

General Features of the Dorsal Horn Neurons

All in all, 180 neurons that responded to the electrical search stimulus were evaluated in the 25 animals of all groups (Table 1). Of these 180 neurons, 144 responded to at least one of the mechanical or chemical stimuli used, whereas 36 did not respond to mechanical stimuli. Just over one-third (35.4%) of the 144 neurons (51/144) received input from deep tissues (muscles, fascia, and other deep soft tissues), and 86.1% of the neurons (124/144) had input from the skin. Many neurons (22.9%, 33/144) had convergent input in that they responded to stimulation of two or more types of tissue (such as skin and muscle/fascia or 2 different kinds of deep tissues). In the whole sample of recorded neurons (n = 180), 10.6% of all neurons (19/180) recorded responded exclusively to stimulation of deep soft tissues.

Table 1.

Input sources and resting activity of dorsal horn neurons

Treatment group PBS NGF Mino-Pre Mino-Post Fluoro-Post All Groups
Number of animals 5 5 5 5 5 25
Number of neurons 37 34 34 37 38 180
Input sources, n
All deep tissues 6 17 6 16 6 51
Low back 3 10 2 7 5 27
Skin 23 21 24 27 29 124
Unknown 11 6 6 6 7 36
Convergent input 3 12 2 12 4 33
Deep receptive fields outside the low back 3 9 4 9 1 26
Resting activity, imp/min
Mean 29.1 100.7 6.4 32.6 9.9
Median 0 1.5 0 1 0
IQR 0 19.5 6 29 6.2
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NGF, nerve growth factor; Mino, minocycline; Fluoro, fluorocitrate; IQR, interquartile range.

Recording Depth

The recording depth of the dorsal horn neurons studied ranged from 100 to 935 µm; there were no significant differences between the five treatment groups. Almost two-thirds (62.8%, 113/180) of the neurons were recorded at a depth of 400–800 µm, which corresponds to laminae IV, V, and VI of the dorsal horn. Almost two-thirds (64.7%, 33/51) of the neurons having input from deep somatic tissues were located in these laminae. Again, no significant difference in recording depth of the neurons having deep somatic input was found between the treatment groups.

Input Source of Dorsal Horn Neurons

Neurons responding to input from deep tissues.

Animals of the NGF group exhibited an increase in the proportion of dorsal horn neurons responding to stimulation of at least one of the deep somatic tissues tested, namely muscles, fascia, and nonidentified soft tissues (Fig. 2A). Compared with the PBS group that received two PBS injections instead of NGF, the increase was statistically significant (P < 0.01). The NGF-induced increase in deep input was significantly reduced when minocycline was given before the first NGF injection (mino-pre group, P < 0.01, Fig. 2A) but not when minocycline was administrated after the injection (mino-post group). Fluorocitrate given after the first NGF injection, however, reduced significantly the input from deep tissues and brought the deep input level back to normal (fluoro-post group, P < 0.01, Fig. 2A).

Two NGF injections significantly unmasked formerly ineffective synaptic connections to the deep tissues in the low back close to the injection site (PBS group vs. NGF group, P < 0.05, Fig. 3B). Compared with the NGF group, the input from the deep tissues in the low back was significantly lower in the mino-pre group when minocycline administration started before NGF injection (P < 0.05, Fig. 3B). Treatment with minocycline and fluorocitrate after the first NGF injection had no significant effect (mino-post and fluoro-post groups). An interesting effect of the two NGF injections into the multifidus muscle was the appearance of new receptive fields in deep tissues outside the injected low back (NGF group, Fig. 3A). Compared with the PBS group in the NGF group, more neurons responded to input from deep tissues outside the low back (P = 0.057). These new receptive fields appeared in the hip and in the hind-limb (gray areas in Fig. 3A). Posttreatment with fluorocitrate (fluoro-post group) reduced this input significantly (P < 0.01, Fig. 3C).

Fig. 3.

Fig. 3.

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Receptive fields located in deep somatic tissues. A: outlines of the rat body showing the approximate location and size of deep receptive fields. Open areas are deep receptive fields located in the low back (multifidus muscle, thoracolumbar fascia); hatched areas are deep receptive fields located outside the low back. B: proportion of neurons with receptive fields in the multifidus muscle and/or the thoracolumbar fascia (open areas in A). C: proportion of neurons with receptive fields in deep tissues outside the low back (hip, entire hind limb; hatched areas in A). B and C are subpopulations of the neurons shown in Fig. 2A. Experimental groups and numbers are in parentheses as in Fig. 2.

Neurons responding to noxious or innocuous stimulation of deep tissues.

After two NGF injections, the proportion of neurons that responded to noxious but not to innocuous mechanical stimulation of deep tissues increased significantly (high-threshold mechanosensitive; PBS group vs. NGF group, P < 0.05, Fig. 4A). Compared with the NGF group, the nociceptive input from the deep tissues was significantly reduced in the mino-pre group (P < 0.01, Fig. 4A) but not mino-post group. Treatment with fluorocitrate after the first NGF injection also reduced the nociceptive input, but the effect was not significant (Fig. 4A, fluoro-post group, P = 0.1).

Fig. 4.

Fig. 4.

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Responsiveness to noxious and innocuous stimulation of deep tissues. A: proportion of neurons that responded to noxious but not to innocuous mechanical stimulation of deep tissues. HTM, high-threshold mechanosensitive. B: neurons that responded to moderate, innocuous pressure applied to deep somatic structures. LTM, low-threshold mechanosensitive. Experimental groups and numbers are in parentheses as in Fig. 2.

The number of neurons that responded to moderate, innocuous pressure applied to deep somatic structures (low-threshold mechanosensitive) likewise increased after two NGF injections, but the increase was not significant (PBS group vs. NGF group, P = 0.098, Fig. 4B). Compared with the NGF group, the proportion of neurons with innocuous deep input was significantly lower when fluorocitrate was administrated (fluoro-post group, P < 0.01, Fig. 4B).

Neurons responding to skin input.

In contrast to neurons having input from deep tissues, the proportion of neurons responding to input from the skin remained unchanged in all treatment groups (Fig. 2B). No significant differences were found between the proportions of neurons processing nociceptive and innocuous skin input.

Neurons having convergent input.

The proportion of neurons having convergent input from various tissues of the low back, hip, and hind-limb (muscle, fascia skin) was markedly increased following two NGF injections (PBS group vs. NGF group, P < 0.01, Fig. 5B). This finding indicates that some neurons must have acquired new receptive fields, i.e., synaptic connections have been unmasked by NGF. As the proportion of neurons having input from the skin remained largely constant in all treatment groups (Fig. 2B), the new receptive fields found after two NGF injections (NGF group) must have been situated in deep somatic tissues. The proportion of convergent neurons was significantly reduced in the mino-pre group (P < 0.01, Fig. 5B) and in the fluoro-post group (P < 0.05, Fig. 5B) but not in the mino-post group.

Fig. 5.

Fig. 5.

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Dorsal horn neurons with convergent input (neurons with input from >1 type of tissue). A: recording from a single neuron (NGF group). a: Responses of the neuron to noxious stimulation applied to the multifidus (MF) muscle (noxious pressure) and to touching or pinching the skin (touch, pinch, skin). Open bars underneath the registrations indicate time and duration of stimulation. b: Outline of the rat body showing the approximate location and size of the receptive fields. Black area shows receptive field in the MF muscle; white area shows receptive field in the skin. B: proportion of neurons with convergent input in the 5 treatment groups. Experimental groups and numbers are in parentheses as in Fig. 2.

All receptive fields were located ipsilateral to the recording site of the dorsal horn neuron. The mean size of deep and cutaneous receptive fields was unchanged in all treatment groups.

Resting (Ongoing) Activity of Dorsal Horn Neurons

Compared with the PBS group, both the proportion of neurons having resting activity (P < 0.01, Fig. 6C) and the discharge frequency (imp/min) across all, discharging and silent, neurons increased significantly after two NGF injections (P < 0.01, Fig. 6B) and also in the mino-post group (both P < 0.01). The NGF-induced increase in discharge frequency was not significantly reduced by intrathecal administration of minocycline or fluorocitrate (Fig. 6B; mean, median, and interquartile range are given in Table 1). However, the discharge frequency in the mino-pre group showed a trend toward lower values (Fig. 6B, Table 1; NGF vs. mino-pre group, P = 0.147).

Fig. 6.

Fig. 6.

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Resting activity of dorsal horn neurons. A: original registrations from 2 active neurons (marked in B). a: Neuron with low resting activity. b: Neuron with high resting activity. B: discharge frequency of all neurons (with and without resting activity). Open arrows indicate the median in each group (see also Table 1). C: proportion of neurons having resting activity. Experimental groups and numbers are in parentheses as in Fig. 2. The P value indicates statistically significant differences between the treatment groups (B: Mann-Whitney U-test; C: Fisher’s exact probability test).

In all treatment groups, the neurons having resting activity were nearly equally distributed over the recording period (Fig. 7). There was no preferred time window after the last injection. Likewise, neurons with a high ongoing discharge frequency showed no preferred time window during the recording period.

Fig. 7.

Fig. 7.

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Time point of measuring the resting activity for active and silent neurons during the 4 h of the recording session. Solid circles, active neurons having receptive fields (RF) in deep tissues of the low back close to the injection site excluding the hip and the thigh; shaded circles, active neurons having RF in tissues other than the low back; open circles, silent neurons (no resting activity). Experimental groups are as in Fig. 2.

DISCUSSION

In the present study, we used two NGF injections into the multifidus muscle at an interval of 5 days as a noninflammatory model of low back pain (Hoheisel et al. 2013). NGF release is closely associated with many painful muscle disorders as well as with nonpainful overloaded muscles (Hayashi et al. 2011; Murase et al. 2010). NGF has been found to excite exclusively nociceptive muscle afferents (Hoheisel et al. 2005), which evoked mainly subthreshold potentials in dorsal horn neurons (Hoheisel et al. 2007). A single NGF injection into the multifidus muscle caused just a short-lasting transient sensitization of dorsal horn neurons, which was followed by a state of latent sensitization (Hoheisel et al. 2013). In this state, the neurons behave normally but could be sensitized for a longer period of time by a subsequent second NGF injection, indicating a priming of the nociceptive system (DeSantana and Sluka 2008; Ferrari et al. 2010; Hendrich et al. 2013; Pereira et al. 2015; Reichling and Levine 2009).

Latent Sensitization and Glial Cell Activation

Blocking the microglia activation by minocycline given before the first NGF injection prevented the NGF-induced state of latent sensitization (mino-pre group). The second NGF injection no longer led to the stronger neuronal sensitization as found in the control group without minocycline administration (NGF group).

An important finding was that fluorocitrate but not minocycline reversed the latent sensitization of dorsal horn neurons. Fluorocitrate given after the first NGF injection significantly reduced the sensitizing effect of the second NGF injection, indicating that the latent neuronal sensitization caused by the first NGF injection was abolished. In contrast, when minocycline was administrated after the first NGF injection, the second NGF injection still showed the strong sensitizing effect, indicating that latent sensitization was not reversed.

The effectiveness of glial inhibitors used in male rats in the present study is in line with their inhibitory effect on neuropathic and inflammatory pain in male mice or rats in studies of other groups (Sorge et al. 2015; Taves et al. 2016). Substances released by glial cells (such as TNF-α, IL-1β) cause a long-lasting hyperexcitability of spinal neurons (Sandkühler and Lee 2013; Svensson et al. 2003; Xanthos and Sandkühler 2014; Yamamoto et al. 2015). Accordingly, the pain-related behavior of animals was markedly attenuated by blocking glial cells (Chacur et al. 2009; Clark et al. 2007; Mei et al. 2011; Milligan et al. 2003; Obata et al. 2006; Raghavendra et al. 2003).

In a neuropathic pain model, the development of mechanical hyperalgesia was significantly reduced by minocycline given before but not after induction of the nerve lesion (Raghavendra et al. 2003). Mei and colleagues (Mei et al. 2011), however, reported a weak but still significant reduction of mechanical hyperalgesia when minocycline was administrated after nerve injury. Other data indicate that, in contrast to mechanical hyperalgesia, only the thermal hyperalgesia was reversed by minocycline (Vanelderen et al. 2013). Fluorocitrate reversed mechanical hyperalgesia in animal models of neuropathic pain (Clark et al. 2007; Milligan et al. 2003; Obata et al. 2006), indicating that astrocyte activation is mainly involved in the maintenance of spinal hypersensitivity. In contrast, fluorocitrate did not reverse an existing hyperalgesia induced by repeated acidic saline injections into the rat gastrocnemius soleus muscle (Ledeboer et al. 2006). However, in this study, fluorocitrate was administrated as a single bolus after the second injection of acidic saline in a state of manifest hyperalgesia.

The data of the present study show that, in the NGF-induced latent sensitization of spinal neurons, having input from deep soft tissues of the low back glial cells plays a similarly important role as in other chronic pain states. Minocycline prevents the latent sensitization induced by NGF but cannot reverse it. In contrast, a block of additional glial cells including astrocytes with fluorocitrate is capable of reversing an existing latent sensitization. Therefore, microglia appears to be responsible for the development of latent sensitization following the first NGF sensitization. For reversal of a spinal sensitization, astrocytes have to be blocked, indicating that the maintenance of latent sensitization depends on astrocyte activation. However, it has to be kept in mind that both inhibitors used were not fully selective, and nonspecific effects cannot be excluded (Clark and Malcangio 2014; Gruber-Schoffnegger et al. 2013, Möller et al. 2016).

A recent doctoral thesis from our laboratory using immunohistochemical techniques suggests that, after two NGF injections into the multifidus muscle, microglia glial cells are activated (Fig. 8; Zhang 2016, dissertation, Heidelberg University).

Fig. 8.

Fig. 8.

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Images of microglial cells, astrocytes, and quantitative analysis. A: microglial cells visualized by ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactivity. a: 1 day after the second PBS injection (see Fig. 1, control). b: 1 day after the second NGF injection. c and d: Quantitative analysis (same region as shown in Aa and Ab). c: Circularity of microglial cells (circularity increases when microglial cells become less ramified). d: Mean number of intersections (Sholl analysis: quantitative study of the radial distribution of the arborization pattern). Open bars are PBS injection; solid bars are NGF injection. Fifteen sections from 5 animals were evaluated, using image analysis software ImageJ (NIH, Bethesda, MD). B: astrocytes visualized by glial fibrillary acidic protein immunoreactivity (a, PBS injections; b, NGF injections). Circularity is as follows: PBS, 0.82 ± 0.013; NGF, 0.83 ± 0.0094, not significant; Sholl analysis: not tested, too much overlap of cells. Scale bars = 30 µm, thickness of the tissue sections = 20 µm. Spinal segment L2, neck of the dorsal horn. Microglial cells showed a plumper form (increased circularity) and lesser ramification (decreased number of intersections) after 2 NGF injections compared with the PBS control group. Changes in astrocyte immunohistochemistry did not reach significant values.

Several parameters like circularity (Zanier et al. 2015; circularity increases when the cells become less ramified, Fig. 8Ac) or Sholl analysis (Ferreira et al. 2014; Morrison and Filosa 2013, quantitative analysis of the radial distribution of the arborization pattern, Fig. 8Ad) indicate that, after NGF, microglial cells were plumper in form (increased circularity) and less ramified (decreased number of intersections) compared with the control situation after PBS injections. Immunohistochemical signs of an activation of astrocytes after NGF were much weaker compared with microglial cells and did not reach significant values.

Appearance of New Receptive Fields

A prominent finding in the animal model used was the appearance of new receptive fields in deep somatic tissues after repeated NGF injection (Figs. 2A and 3A; Hoheisel et al. 2013). Most of the new deep receptive fields were located outside the low back (injection site of NGF) and extended into the distal hind-limb (Fig. 3, A and C). One explanation for this result is the opening of silent or sleeping synaptic connections by the NGF-induced afferent input. This means that anatomically existing but functionally silent connections were unmasked so that they now could excite the neuron (Wall 1988). Comparable changes in the responsiveness of dorsal horn neurons have been reported in animal models of inflammatory pain. In animals with an inflamed fascia or inflamed knee joint, the receptive fields expanded into healthy tissues adjacent to the inflamed region (Hoheisel and Mense 2015; Neugebauer et al. 1993; Neugebauer and Schaible 1990).

In the present study, no new receptive field appeared after the second NGF injection when the state of latent sensitization in dorsal horn neurons had been prevented or reversed by blocking glial cell activation. One interpretation is that glial cells activated by the NGF-induced input are important for the synaptic opening process, e.g., via fractalkine released from active primary afferent fibers (Clark and Malcangio 2014; Owolabi and Saab 2006; Xanthos and Sandkühler 2014). Spinal terminals of active primary afferent fibers release fractalkine, which binds to the CX3CR1 receptor expressed primarily on microglia (Ali et al. 2015; Xanthos and Sandkühler 2014). Spinal fractalkine application leads to a long-lasting increase in the C fiber- induced spinal synaptic field potential, indicating a long-term potentiation of the synaptic strength (Clark et al. 2015). This synaptic long-term potentiation may be blocked by prophylactic minocycline application (Clark et al. 2015).

Resting Activity of Dorsal Horn Neurons

Spontaneous pain and dysesthesia are probably due to the resting activity in nociceptive neurons of the central nervous system, including the spinal cord (Djouhri et al. 2006; Isnard et al. 2011; Suzuki and Dickenson 2006). In the present experiments, the dorsal horn neurons exhibited a significantly higher resting activity when two NGF injections were made into the multifidus muscle. The NGF-induced increase in resting activity was not influenced by minocycline or by fluorocitrate.

This finding indicates that the responsiveness of the neurons to peripheral input and the neuronal resting activity are controlled by different mechanisms. The neuronal responsiveness appears to be dependent on glia activation, whereas the resting activity seems to be controlled by other mechanisms independent of glia activation. Chronic stress, for example, promotes low back pain (Mendelek et al. 2013) and increases resting activity of the dorsal horn neurons, whereas it has little effect on their responsiveness to external stimuli (Hoheisel et al. 2015).

Technical Considerations

In the present study, fluorocitrate has been administered 2 days after the first NGF injection but not like minocycline before the injection of the growth factor. Minocycline significantly prevented the NGF-induced neuronal sensitization, and it is likely that fluorocitrate, as an unspecific inhibitor of glial cell activation, injected at the same time would have also blocked microglia and prevented dorsal horn sensitization. This possibility has not been tested though because we wanted to spare the lives of additional animals. Therefore, we cannot exclude that astrocytes may have also contributed to the early phase of latent sensitization. We just administered fluorocitrate after the first NGF to see whether fluorocitrate, in contrast to minocycline, was capable of reversing the neuronal sensitization.

The implanted osmotic pump and the intrathecal catheter inserted contralateral to the NGF injection site between vertebrae L5 and L6 alone did not affect the behavior of the recorded dorsal horn neurons. In the present study, nearly the same NGF-induced changes were observed (comparing the PBS and the NGF group) as described in a previous study of our group made of “intact” animals without an intrathecal catheter (Hoheisel et al. 2013). Both data sets are largely identical, indicating that the dorsal horn neurons at the recording level were not sensitized by the surgery or the intrathecal catheter.

The experimenters were not blinded. However, to minimize the danger of experimenter bias, the search for receptive fields followed a fixed protocol that was strictly used in all treatment groups.

Conclusions

Collectively, the data suggest that activation of glial cells plays an important role in the latent sensitization of spinal dorsal horn neurons caused by NGF-induced muscle input. Especially the state of latent sensitization, in which dorsal horn neurons behave normally but can be sensitized for longer periods of time by a subsequent nociceptive input, is mediated by activated glial cells. Spinal microglia appear to control the development of the neuronal sensitization but not its maintenance. Astrocytes are more involved in its maintenance; their possible contribution to induction was not tested. The effects in our latent sensitization model of low back pain were similar to neuropathic and inflammatory pain models.

GRANTS

This work was funded by grants from the German Federal Ministry of Education and Research (BMBF, 01EC1010B) and the Deutsche Forschungsgemeinschaft (DGF, TR 236/24-1, ME 492/16-1 and SFB 1158, project S01).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.M., R.-D.T., and U.H. conceived and designed experiments; J.Z. performed experiments; J.Z. analyzed data; J.Z., S.M., R.-D.T., and U.H. interpreted results of experiments; J.Z. prepared figures; J.Z. and U.H. drafted manuscript; J.Z., S.M., R.-D.T., and U.H. edited and revised manuscript; J.Z., S.M., R.-D.T., and U.H. approved final version of manuscript.

ACKNOWLEDGMENTS

The authors thank E. Hofmann for excellent technical assistance and the financial support of the China Scholarship Council (CSC).

REFERENCES

  1. Ali I, Chugh D, Ekdahl CT. Role of fractalkine-CX3CR1 pathway in seizure-induced microglial activation, neurodegeneration, and neuroblast production in the adult rat brain. Neurobiol Dis 74: 194–203, 2015. doi: 10.1016/j.nbd.2014.11.009. [DOI] [PubMed] [Google Scholar]
  2. Balagué F, Mannion AF, Pellisé F, Cedraschi C. Nonspecific low back pain. Lancet 379: 482–491, 2012. doi: 10.1016/S0140-6736(11)60610-7. [DOI] [PubMed] [Google Scholar]
  3. Cao J, Li Z, Zhang Z, Ren X, Zhao Q, Shao J, Li M, Wang J, Huang P, Zang W. Intrathecal injection of fluorocitric acid inhibits the activation of glial cells causing reduced mirror pain in rats. BMC Anesthesiol 14: 119, 2014. doi: 10.1186/1471-2253-14-119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chacur M, Lambertz D, Hoheisel U, Mense S. Role of spinal microglia in myositis-induced central sensitisation: an immunohistochemical and behavioural study in rats. Eur J Pain 13: 915–923, 2009. doi: 10.1016/j.ejpain.2008.11.008. [DOI] [PubMed] [Google Scholar]
  5. Clark AK, Gentry C, Bradbury EJ, McMahon SB, Malcangio M. Role of spinal microglia in rat models of peripheral nerve injury and inflammation. Eur J Pain 11: 223–230, 2007. doi: 10.1016/j.ejpain.2006.02.003. [DOI] [PubMed] [Google Scholar]
  6. Clark AK, Gruber-Schoffnegger D, Drdla-Schutting R, Gerhold KJ, Malcangio M, Sandkühler J. Selective activation of microglia facilitates synaptic strength. J Neurosci 35: 4552–4570, 2015. doi: 10.1523/JNEUROSCI.2061-14.2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clark AK, Malcangio M. Fractalkine/CX3CR1 signaling during neuropathic pain. Front Cell Neurosci 8: 121, 2014. doi: 10.3389/fncel.2014.00121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Coull JA, Beggs S, Boudreau D, Boivin D, Tsuda M, Inoue K, Gravel C, Salter MW, De Koninck Y. BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature 438: 1017–1021, 2005. doi: 10.1038/nature04223. [DOI] [PubMed] [Google Scholar]
  9. Deising S, Weinkauf B, Blunk J, Obreja O, Schmelz M, Rukwied R. NGF-evoked sensitization of muscle fascia nociceptors in humans. Pain 153: 1673–1679, 2012. doi: 10.1016/j.pain.2012.04.033. [DOI] [PubMed] [Google Scholar]
  10. DeSantana JM, Sluka KA. Central mechanisms in the maintenance of chronic widespread noninflammatory muscle pain. Curr Pain Headache Rep 12: 338–343, 2008. doi: 10.1007/s11916-008-0057-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Djouhri L, Koutsikou S, Fang X, McMullan S, Lawson SN. Spontaneous pain, both neuropathic and inflammatory, is related to frequency of spontaneous firing in intact C-fiber nociceptors. J Neurosci 26: 1281–1292, 2006. doi: 10.1523/JNEUROSCI.3388-05.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ferrari LF, Bogen O, Levine JD. Nociceptor subpopulations involved in hyperalgesic priming. Neuroscience 165: 896–901, 2010. doi: 10.1016/j.neuroscience.2009.11.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ferreira TA, Blackman AV, Oyrer J, Jayabal S, Chung AJ, Watt AJ, Sjöström PJ, van Meyel DJ. Neuronal morphometry directly from bitmap images. Nat Methods 11: 982–984, 2014. doi: 10.1038/nmeth.3125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gerhold KJ, Drdla-Schutting R, Honsek SD, Forsthuber L, Sandkühler J. Pronociceptive and antinociceptive effects of buprenorphine in the spinal cord dorsal horn cover a dose range of four orders of magnitude. J Neurosci 35: 9580–9594, 2015. doi: 10.1523/JNEUROSCI.0731-14.2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gibson W, Arendt-Nielsen L, Taguchi T, Mizumura K, Graven-Nielsen T. Increased pain from muscle fascia following eccentric exercise: animal and human findings. Exp Brain Res 194: 299–308, 2009. doi: 10.1007/s00221-008-1699-8. [DOI] [PubMed] [Google Scholar]
  16. Gruber-Schoffnegger D, Drdla-Schutting R, Hönigsperger C, Wunderbaldinger G, Gassner M, Sandkühler J. Induction of thermal hyperalgesia and synaptic long-term potentiation in the spinal cord lamina I by TNF-α and IL-1β is mediated by glial cells. J Neurosci 33: 6540–6551, 2013. doi: 10.1523/JNEUROSCI.5087-12.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hayashi K, Ozaki N, Kawakita K, Itoh K, Mizumura K, Furukawa K, Yasui M, Hori K, Yi S-Q, Yamaguchi T, Sugiura Y. Involvement of NGF in the rat model of persistent muscle pain associated with taut band. J Pain 12: 1059–1068, 2011. doi: 10.1016/j.jpain.2011.04.010. [DOI] [PubMed] [Google Scholar]
  18. Hendrich J, Alvarez P, Joseph EK, Chen X, Bogen O, Levine JD. Electrophysiological correlates of hyperalgesic priming in vitro and in vivo. Pain 154: 2207–2215, 2013. doi: 10.1016/j.pain.2013.07.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hoheisel U, Mense S. Inflammation of the thoracolumbar fascia excites and sensitizes rat dorsal horn neurons. Eur J Pain 19: 419–428, 2015. doi: 10.1002/ejp.563. [DOI] [PubMed] [Google Scholar]
  20. Hoheisel U, Reuter R, de Freitas MF, Treede RD, Mense S. Injection of nerve growth factor into a low back muscle induces long-lasting latent hypersensitivity in rat dorsal horn neurons. Pain 154: 1953–1960, 2013. doi: 10.1016/j.pain.2013.05.006. [DOI] [PubMed] [Google Scholar]
  21. Hoheisel U, Taguchi T, Treede RD, Mense S. Nociceptive input from the rat thoracolumbar fascia to lumbar dorsal horn neurones. Eur J Pain 15: 810–815, 2011. doi: 10.1016/j.ejpain.2011.01.007. [DOI] [PubMed] [Google Scholar]
  22. Hoheisel U, Unger T, Mense S. Excitatory and modulatory effects of inflammatory cytokines and neurotrophins on mechanosensitive group IV muscle afferents in the rat. Pain 114: 168–176, 2005. doi: 10.1016/j.pain.2004.12.020. [DOI] [PubMed] [Google Scholar]
  23. Hoheisel U, Unger T, Mense S. Sensitization of rat dorsal horn neurons by NGF-induced subthreshold potentials and low-frequency activation. A study employing intracellular recordings in vivo. Brain Res 1169: 34–43, 2007. doi: 10.1016/j.brainres.2007.06.054. [DOI] [PubMed] [Google Scholar]
  24. Hoheisel U, Vogt MA, Palme R, Gass P, Mense S. Immobilization stress sensitizes rat dorsal horn neurons having input from the low back. Eur J Pain 19: 861–870, 2015. doi: 10.1002/ejp.682. [DOI] [PubMed] [Google Scholar]
  25. Isnard J, Magnin M, Jung J, Mauguière F, Garcia-Larrea L. Does the insula tell our brain that we are in pain? Pain 152: 946–951, 2011. doi: 10.1016/j.pain.2010.12.025. [DOI] [PubMed] [Google Scholar]
  26. Ledeboer A, Mahoney JH, Milligan ED, Martin D, Maier SF, Watkins LR. Spinal cord glia and interleukin-1 do not appear to mediate persistent allodynia induced by intramuscular acidic saline in rats. J Pain 7: 757–767, 2006. doi: 10.1016/j.jpain.2006.04.001. [DOI] [PubMed] [Google Scholar]
  27. Ledeboer A, Sloane EM, Milligan ED, Frank MG, Mahony JH, Maier SF, Watkins LR. Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation. Pain 115: 71–83, 2005. doi: 10.1016/j.pain.2005.02.009. [DOI] [PubMed] [Google Scholar]
  28. Malcangio M. Microglia and chronic pain. Pain 157: 1002–1003, 2016. doi: 10.1097/j.pain.0000000000000509. [DOI] [PubMed] [Google Scholar]
  29. McMahon SB, Malcangio M. Current challenges in glia-pain biology. Neuron 64: 46–54, 2009. doi: 10.1016/j.neuron.2009.09.033. [DOI] [PubMed] [Google Scholar]
  30. Mei XP, Xu H, Xie C, Ren J, Zhou Y, Zhang H, Xu LX. Post-injury administration of minocycline: an effective treatment for nerve-injury induced neuropathic pain. Neurosci Res 70: 305–312, 2011. doi: 10.1016/j.neures.2011.03.012. [DOI] [PubMed] [Google Scholar]
  31. Mendelek F, Caby I, Pelayo P, Kheir RB. The application of a classification-tree model for predicting low back pain prevalence among hospital staff. Arch Environ Occup Health 68: 135–144, 2013. doi: 10.1080/19338244.2012.663010. [DOI] [PubMed] [Google Scholar]
  32. Milligan ED, Twining C, Chacur M, Biedenkapp J, O’Connor K, Poole S, Tracey K, Martin D, Maier SF, Watkins LR. Spinal glia and proinflammatory cytokines mediate mirror-image neuropathic pain in rats. J Neurosci 23: 1026–1040, 2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Milligan ED, Watkins LR. Pathological and protective roles of glia in chronic pain. Nat Rev Neurosci 10: 23–36, 2009. doi: 10.1038/nrn2533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mogil JS, Davis KD, Derbyshire SW. The necessity of animal models in pain research. Pain 151: 12–17, 2010. doi: 10.1016/j.pain.2010.07.015. [DOI] [PubMed] [Google Scholar]
  35. Möller T, Bard F, Bhattacharya A, Biber K, Campbell B, Dale E, Eder C, Gan L, Garden GA, Hughes ZA, Pearse DD, Staal RG, Sayed FA, Wes PD, Boddeke HW. Critical data-based re-evaluation of minocycline as a putative specific microglia inhibitor. Glia 64: 1788–1794, 2016. doi: 10.1002/glia.23007. [DOI] [PubMed] [Google Scholar]
  36. Morrison HW, Filosa JA. A quantitative spatiotemporal analysis of microglia morphology during ischemic stroke and reperfusion. J Neuroinflamm 10: 4, 2013. doi: 10.1186/1742-2094-10-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Murase S, Terazawa E, Queme F, Ota H, Matsuda T, Hirate K, Kozaki Y, Katanosaka K, Taguchi T, Urai H, Mizumura K. Bradykinin and nerve growth factor play pivotal roles in muscular mechanical hyperalgesia after exercise (delayed-onset muscle soreness). J Neurosci 30: 3752–3761, 2010. doi: 10.1523/JNEUROSCI.3803-09.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Neugebauer V, Lücke T, Schaible HG. N-methyl-d-aspartate (NMDA) and non-NMDA receptor antagonists block the hyperexcitability of dorsal horn neurons during development of acute arthritis in rat’s knee joint. J Neurophysiol 70: 1365–1377, 1993. [DOI] [PubMed] [Google Scholar]
  39. Neugebauer V, Schaible HG. Evidence for a central component in the sensitization of spinal neurons with joint input during development of acute arthritis in cat’s knee. J Neurophysiol 64: 299–311, 1990. [DOI] [PubMed] [Google Scholar]
  40. Obata H, Eisenach JC, Hussain H, Bynum T, Vincler M. Spinal glial activation contributes to postoperative mechanical hypersensitivity in the rat. J Pain 7: 816–822, 2006. doi: 10.1016/j.jpain.2006.04.004. [DOI] [PubMed] [Google Scholar]
  41. Owolabi SA, Saab CY. Fractalkine and minocycline alter neuronal activity in the spinal cord dorsal horn. FEBS Lett 580: 4306–4310, 2006. doi: 10.1016/j.febslet.2006.06.087. [DOI] [PubMed] [Google Scholar]
  42. Pereira MP, Donahue RR, Dahl JB, Werner M, Taylor BK, Werner MU. Endogenous opioid-masked latent pain sensitization: studies from mouse to human. PLoS One 10: e0134441, 2015. doi: 10.1371/journal.pone.0134441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Philippu A. Use of push-pull cannulae to determine the release of endogenous neurotransmitters in distinct brain areas of anaesthetized and freely moving animals. Measurement of neurotransmitter release in vivo. In: IBRO Handbook Series: Methods in the Neuroscience, vol. 6, edited by Marsden CA. New York, NY: Wiley, 1984, pp. 3–37. [Google Scholar]
  44. Raghavendra V, Tanga F, DeLeo JA. Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy. J Pharmacol Exp Ther 306: 624–630, 2003. doi: 10.1124/jpet.103.052407. [DOI] [PubMed] [Google Scholar]
  45. Raghavendra V, Tanga FY, DeLeo JA. Complete Freunds adjuvant-induced peripheral inflammation evokes glial activation and proinflammatory cytokine expression in the CNS. Eur J Neurosci 20: 467–473, 2004. doi: 10.1111/j.1460-9568.2004.03514.x. [DOI] [PubMed] [Google Scholar]
  46. Reichling DB, Levine JD. Critical role of nociceptor plasticity in chronic pain. Trends Neurosci 32: 611–618, 2009. doi: 10.1016/j.tins.2009.07.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sandkühler J, Lee J. How to erase memory traces of pain and fear. Trends Neurosci 36: 343–352, 2013. doi: 10.1016/j.tins.2013.03.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schilder A, Hoheisel U, Magerl W, Benrath J, Klein T, Treede RD. Sensory findings after stimulation of the thoracolumbar fascia with hypertonic saline suggest its contribution to low back pain. Pain 155: 222–231, 2014. doi: 10.1016/j.pain.2013.09.025. [DOI] [PubMed] [Google Scholar]
  49. Schilder A, Magerl W, Hoheisel U, Klein T, Treede RD. Electrical high-frequency stimulation of the human thoracolumbar fascia evokes long-term potentiation-like pain amplification. Pain 157: 2309–2317, 2016. doi: 10.1097/j.pain.0000000000000649. [DOI] [PubMed] [Google Scholar]
  50. Sorge RE, Mapplebeck JCS, Rosen S, Beggs S, Taves S, Alexander JK, Martin LJ, Austin JS, Sotocinal SG, Chen D, Yang M, Shi XQ, Huang H, Pillon NJ, Bilan PJ, Tu Y, Klip A, Ji RR, Zhang J, Salter MW, Mogil JS. Different immune cells mediate mechanical pain hypersensitivity in male and female mice. Nat Neurosci 18: 1081–1083, 2015. doi: 10.1038/nn.4053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Stecco C, Stern R, Porzionato A, Macchi V, Masiero S, Stecco A, De Caro R. Hyaluronan within fascia in the etiology of myofascial pain. Surg Radiol Anat 33: 891–896, 2011. doi: 10.1007/s00276-011-0876-9. [DOI] [PubMed] [Google Scholar]
  52. Sung CS, Cherng CH, Wen ZH, Chang WK, Huang SY, Lin SL, Chan KH, Wong CS. Minocycline and fluorocitrate suppress spinal nociceptive signaling in intrathecal IL-1β-induced thermal hyperalgesic rats. Glia 60: 2004–2017, 2012. doi: 10.1002/glia.22415. [DOI] [PubMed] [Google Scholar]
  53. Suzuki R, Dickenson AH. Differential pharmacological modulation of the spontaneous stimulus-independent activity in the rat spinal cord following peripheral nerve injury. Exp Neurol 198: 72–80, 2006. doi: 10.1016/j.expneurol.2005.10.032. [DOI] [PubMed] [Google Scholar]
  54. Svensson P, Cairns BE, Wang K, Arendt-Nielsen L. Injection of nerve growth factor into human masseter muscle evokes long-lasting mechanical allodynia and hyperalgesia. Pain 104: 241–247, 2003. doi: 10.1016/S0304-3959(03)00012-5. [DOI] [PubMed] [Google Scholar]
  55. Taguchi T, Hoheisel U, Mense S. Dorsal horn neurons having input from low back structures in rats. Pain 138: 119–129, 2008. doi: 10.1016/j.pain.2007.11.015. [DOI] [PubMed] [Google Scholar]
  56. Taves S, Berta T, Liu DL, Gan S, Chen G, Kim YH, Van de Ven T, Laufer S, Ji RR. Spinal inhibition of p38 MAP kinase reduces inflammatory and neuropathic pain in male but not female mice: Sex-dependent microglial signaling in the spinal cord. Brain Behav Immun 55: 70–81, 2016. doi: 10.1016/j.bbi.2015.10.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tikka TM, Koistinaho JE. Minocycline provides neuroprotection against N-methyl-D-aspartate neurotoxicity by inhibiting microglia. J Immunol 166: 7527–7533, 2001. doi: 10.4049/jimmunol.166.12.7527. [DOI] [PubMed] [Google Scholar]
  58. Tsuda M, Masuda T, Tozaki-Saitoh H, Inoue K. Microglial regulation of neuropathic pain. J Pharmacol Sci 121: 89–94, 2013. doi: 10.1254/jphs.12R14CP. [DOI] [PubMed] [Google Scholar]
  59. Vanelderen P, Rouwette T, Kozicz T, Heylen R, Van Zundert J, Roubos EW, Vissers K. Effects of chronic administration of amitriptyline, gabapentin and minocycline on spinal brain-derived neurotrophic factor expression and neuropathic pain behavior in a rat chronic constriction injury model. Reg Anesth Pain Med 38: 124–130, 2013. doi: 10.1097/AAP.0b013e31827d611b. [DOI] [PubMed] [Google Scholar]
  60. Wall PD. Recruitment of ineffective synapses after injury. Adv Neurol 47: 387–400, 1988. [PubMed] [Google Scholar]
  61. Weinkauf B, Deising S, Obreja O, Hoheisel U, Mense S, Schmelz M, Rukwied R. Comparison of nerve growth factor-induced sensitization pattern in lumbar and tibial muscle and fascia. Muscle Nerve 52: 265–272, 2015. doi: 10.1002/mus.24537. [DOI] [PubMed] [Google Scholar]
  62. Xanthos DN, Sandkühler J. Neurogenic neuroinflammation: inflammatory CNS reactions in response to neuronal activity. Nat Rev Neurosci 15: 43–53, 2014. doi: 10.1038/nrn3617. [DOI] [PubMed] [Google Scholar]
  63. Yamamoto Y, Terayama R, Kishimoto N, Maruhama K, Mizutani M, Iida S, Sugimoto T. Activated microglia contribute to convergent nociceptive inputs to spinal dorsal horn neurons and the development of neuropathic pain. Neurochem Res 40: 1000–1012, 2015. doi: 10.1007/s11064-015-1555-8. [DOI] [PubMed] [Google Scholar]
  64. Zanier ER, Fumagalli S, Perego C, Pischiutta F, De Simoni MG. Shape descriptors of the “never resting” microglia in three different acute brain injury models in mice. Intensive Care Med Exp 3: 39, 2015. doi: 10.1186/s40635-015-0039-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zhang J. Prevention and Reversal of Neuronal Sensitization in the Spinal Cord by Blocking Spinal Glial Cell Activation in an Animal Model of Non-Specific Chronic Low Back Pain (PhD Dissertation) Mannheim, Germany: Medical Faculty Mannheim, Heidelberg University, 2016. [Google Scholar]
  66. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16: 109–110, 1983. doi: 10.1016/0304-3959(83)90201-4. [DOI] [PubMed] [Google Scholar]

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