| Evidence Sentence: |
This finding suggested that it might be possible to inhibit nociception for a longer period of time by injecting prostatic acid phosphatase (PAP, ACPP) into acupuncture points. |
| Evidence Sentence: |
To test this, we injected hPAP protein into the popliteal fossa:a novel approach we call PAPupuncture:then monitored noxious thermal and mechanical sensitivity in the hindpaw. |
| Evidence Sentence: |
Strikingly, injection of hPAP (250 mU) into the popliteal fossa inhibited thermal sensitivity for three days only in the injected leg of wild-type mice (Figure 2A). |
| Evidence Sentence: |
Importantly, hPAP did not affect thermal sensitivity in A1R-/- mice, demonstrating a critical requirement for A1R activation (Figure 2A). |
| Evidence Sentence: |
In addition, hPAP did not affect mechanical sensitivity in naive mice (Figure 2B). |
| Evidence Sentence: |
Increased withdrawal latency in the thermal assay was not due to motor impairment, as the same dose of hPAP did not affect motor function in the rotarod test (Figure 2C). |
| Evidence Sentence: |
These results are the first to reveal that a single, peripheral injection of hPAP locally inhibits nociception for an extended period of time by engaging an A1R-dependent mechanism that is common to acupuncture. |
| Evidence Sentence: |
Since hPAP generates adenosine by dephosphorylating AMP, we next sought to determine if the magnitude of the antinociceptive effect could be increased by injecting additional substrate. |
| Evidence Sentence: |
To test this possibility, we injected wild-type mice with hPAP (250 mU) then injected these same mice with AMP (200 nmol) twenty-four hours later. |
| Evidence Sentence: |
AMP injection into the popliteal fossa transiently boosted the magnitude of the antinociceptive effect only in mice previously injected with hPAP (Figure 3A,B). |
| Evidence Sentence: |
To determine if the long-lasting antinociceptive effects of hPAP were due to sustained A1R activation, we next injected hPAP (250 mU) into the popliteal fossa of wild-type mice, then 48 h later injected (i.p.) |
| Evidence Sentence: |
CPX, but not vehicle, transiently blocked the thermal antinociceptive effect of hPAP for four hours (Figure 3C,D). |
| Evidence Sentence: |
In contrast, neither vehicle nor systemic CPX affected thermal sensitivity in the contralateral (non-hPAP injected) hindpaw. |
| Evidence Sentence: |
These data indicate the local antinociceptive effects of hPAP are due to sustained A1R activation. |
| Evidence Sentence: |
We previously found that the antinociceptive effects of hPAP, when injected intrathecally, were due to A1R activation and phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. |
| Evidence Sentence: |
To determine if the antinociceptive effects of PAPupuncture were PLC-dependent, we injected hPAP (250 mU) then 48 h later injected vehicle or U73122 (5.4 nmol) into the ipsilateral popliteal fossa. |
| Evidence Sentence: |
U73122, but not vehicle, transiently blocked the thermal antinociceptive effect of hPAP for four hours (Figure 3E,F). |
| Evidence Sentence: |
Thus, hPAP inhibits nociception through a PLC-dependent mechanism when injected peripherally. |
| Evidence Sentence: |
To test this, we inflamed one hindpaw with complete Freund's adjuvant (CFA) then injected hPAP (250 mU) one day later into the ipsilateral popliteal fossa. |
| Evidence Sentence: |
Likewise, in the spared nerve injury (SNI) model of neuropathic pain, hPAP (250 mU) inhibited thermal and mechanical hypersensitivity for three days in the ipsilateral (injured) hindpaw of wild-type mice but not A1R-/- mice (Figure 4C,D). |
| Evidence Sentence: |
In both chronic pain models, ipsilateral hPAP injections had no effects in the contralateral (non-inflamed/non-injured) hindpaw (Figure 4A-D). |
| Evidence Sentence: |
Collectively, these data indicate that hPAP has localized, long-lasting, A1R-dependent antinociceptive effects in two models of chronic pain when injected peripherally. |
| Evidence Sentence: |
To determine if peripheral injection of hPAP could preemptively inhibit pain hypersensitivity, as occurs when hPAP is injected intrathecally, we injected hPAP (250 mU) into the popliteal fossa of wild-type and A1R-/- mice and performed the SNI surgery the following day. |
| Evidence Sentence: |
hPAP reduced nerve-injury induced thermal hyperalgesia for two days in wild-type but not A1R-/- mice (Figure 4E). |
| Evidence Sentence: |
In addition, injection of hPAP prior to nerve injury reduced mechanical allodynia for six days in wild-type mice (Figure 4F), three days longer than when hPAP was injected after nerve injury (Figure 4D). |
| Evidence Sentence: |
However, given that hPAP did not enduringly reduce hyperalgesia and allodynia for >7 d as hPAP did when injected intrathecally, we conclude that hPAP does not have preemptive antinociceptive effects when administered peripherally. |
| Evidence Sentence: |
There are several possible reasons why hPAP (250 mU) consistently had antinociceptive effects that lasted three days but not longer: 1) hPAP depleted the local AMP pool, 2) A1R desensitized or 3) hPAP protein lost catalytic activity or washed out. |
| Evidence Sentence: |
To distinguish between these possibilities, we injected wild-type mice and A1R-/- mice with hPAP (250 mU) then injected these same mice with a second dose (250 mU) three days later. |
| Evidence Sentence: |
We reasoned that the second hPAP injection should have no antinociceptive effects if the first hPAP injection depleted AMP or desensitized A1R. |
| Evidence Sentence: |
On the contrary, thermal withdrawal latency increased in the ipsilateral hindpaw after the second hPAP injection and remained significantly elevated for four more days in wild-type mice but not A1R-/- mice (Figure 5A). |
| Evidence Sentence: |
These sequential hPAP injections did not affect mechanical sensitivity in naive mice (Figure 5B). |
| Evidence Sentence: |
However, sequential hPAP injections (250 mU on days 21 and 24 post SNI in the ipsilateral popliteal fossa) had a statistically significant antiallodynic effect that lasted for five days (data not shown). |
| Evidence Sentence: |
Since hPAP availability was the limiting factor, we next hypothesized that larger quantities of hPAP might have greater and longer-lasting effects on nociception. |
| Evidence Sentence: |
Indeed, hPAP dose-dependently inhibited the magnitude and duration of noxious thermal sensitivity (Figure 5C,D), with the highest dose (10,000 mU, single injection into popliteal fossa) lasting for six days:nearly 100x longer than acupuncture (which lasts up to 1.5 hr, Figure 6A). |
| Evidence Sentence: |
PAPupuncture antinociception was delayed by 1-6 hr (depending on hPAP dose; Figure 5C), consistent with hPAP working through an enzymatic process and not a pharmacological process (contrast with rapid onset following CPA injection; Figure 1B). |
| Evidence Sentence: |
This finding suggested that it might be possible to inhibit nociception for a longer period of time by injecting prostatic acid phosphatase (PAP, ACPP) into acupuncture points. |
| Evidence Sentence: |
PAP is an ectonucleotidase that dephosphorylates extracellular AMP to adenosine, has a long half-life in vivo and is endogenously found in muscle tissue surrounding acupuncture points. |
| Evidence Sentence: |
Here, we found that injection of PAP into the popliteal fossa:a space behind the knee that encompasses the Weizhong acupuncture point:had dose- and A1R-dependent antinociceptive effects in mouse models of acute and chronic pain. |
| Evidence Sentence: |
Given our previous work with PAP:an ectonucleotidase that hydrolyzes AMP to adenosine and activates A1R for days following a single spinal injection :we hypothesized it might be possible to enzymatically hydrolyze the pool of AMP to adenosine and inhibit nociception without performing acupuncture. |
