Neuromyelitis optica pathology in rats following intraperitoneal injection of NMO-IgG and intracerebral needle injury

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system that can produce motor and visual impairment [1‐3]. Most NMO patients are seropositive for immunoglobulin G autoantibodies (NMO-IgG) directed against aquaporin-4 (AQP4) [4, 5], a water channel expressed in the plasma membrane of astrocytes in brain, spinal cord and optic nerve [6, 7]. Though AQP4 is also expressed in some peripheral tissues, including kidney collecting duct, gastric glands, airway epithelia and skeletal muscle [8, 9], significant pathology is absent in peripheral tissues in NMO [3]. NMO lesions in the human central nervous system show astrocyte damage with loss of AQP4 and glial fibrillary acidic protein (GFAP), inflammation with granulocyte and macrophage infiltration, vasculocentric deposition of activated complement, blood–brain barrier disruption and demyelination [4, 5, 10‐12]. There is a substantial body of evidence supporting a pathogenesis mechanism in which NMO-IgG binding to astrocytic AQP4 produces complement-dependent cytotoxicity (CDC), which leads to inflammation and blood–brain barrier disruption with secondary oligodendrocyte injury, demyelination and neuronal injury [13‐15]. Antibody-dependent cellular cytotoxicity (ADCC) also plays a role [16], as does, perhaps, AQP4-sensitized T cells or other factors promoting blood–brain barrier breakdown [17‐19].

There is considerable interest in creating animal models of NMO for investigation of disease pathogenesis mechanisms and testing therapeutics [20, 21]. The original animal models involved intraperitoneal injection of IgG purified from NMO patient serum in rats with pre-existing inflammation produced by sensitization to myelin oligodendrocyte protein (experimental autoimmune encephalomyelitis, EAE) [22‐24] or by complete Freund’s adjuvant [25]. In these models greater CNS inflammation was seen in rats receiving NMO-IgG, with evidence for astrocyte damage and complement activation. However, the pre-existing inflammation in these models confounds data interpretation because NMO involves astrocyte-targeted antibodies rather than sensitized T cells.

Mouse models of NMO involving intracerebral injection or infusion of NMO-IgG and human complement have been informative in studying disease pathogenesis mechanisms, such as the roles of ADCC [26, 16] and of various leukocyte types [27‐29]. Brain pathology in injected mice is similar to NMO pathology in humans, with loss of AQP4, GFAP and myelin, granulocyte and macrophage infiltration, and complement deposition [19]. In recent advances, including the use of NMO superantibodies with increased CDC/ADCC effector function(s) and CD59 knockout mice, optic neuritis [30] and longitudinally extensive transverse myelitis [31] have been produced in mice by passive transfer of NMO-IgG and human complement. However, all mouse models require direct administration of human complement into the central nervous system, as the mouse complement system is ineffective because, in part, of circulating complement-inactivating protein(s) [32].

To overcome the limitations of existing models, and building on methods developed in mice, we recently reported a rat model of NMO involving intracerebral injection of NMO-IgG, without complement supplementation and without pre-existing neuroinflammation [33]. Unlike mice, rats have an active complement system similar to humans. The NMO-IgG injected rats developed characteristic NMO pathology around the needle track, which was complement-dependent. The model was applied to investigate the role of ADCC and macrophages in NMO pathogenesis.

A limitation of our reported rat model [33] was the need to inject NMO-IgG directly into the brain, which is different from human NMO in which NMO-IgG is present in serum and pathology is initiated, in large part, following NMO-IgG entry into the central nervous system. Testing of certain therapeutics, such as aquaporumab antibodies [34], IgG inactivation therapies [35, 36] and complement-targeted drugs [37, 38], are best done in models of NMO in which pathology is produced in seropositive animals. Motivated by this need, the goal of this study was to establish a robust of NMO in rats made NMO-IgG-seropositive by peripheral NMO-IgG administration. We first studied the tissue distribution and serum pharmacokinetics of peripherally administered NMO-IgG in rats, and then established the minimal conditions in which robust NMO pathology could be produced in seropositive rats.

The goal of this work was to develop a minimally intrusive, robust model of NMO in NMO-IgG seropositive rats. Pharmacokinetic analysis showed that human IgG was effectively absorbed in rats following IP injection, and that the t_1/2 for NMO-IgG disappearance in blood was ~48 hours. A convenient NMO-IgG dosing regimen was established to maintain rat seropositivity at a level at least as high as that in typical seropositive NMO patients, as verified by a serum cytotoxicity bioassay. Though intravenous NMO-IgG administration could also be used to maintain seropositivity in rats, IP administration is technically easier, particularly when many rats are studied and more than one injection per rat is needed. NMO-IgG administration by mini-pump, though not tested, might also be suitable if subcutaneously delivered antibody is efficiently absorbed.

Tissue distribution studies showed rapid binding of IP-administered NMO-IgG to peripheral AQP4-expressing cells in kidney, stomach, trachea and skeletal muscle, as was found previously in NMO-IgG-injected mice [43]. NMO-IgG was also seen in AQP4-expressing cells in the area postrema of brain, which lacks a blood–brain barrier, but not elsewhere in the central nervous system including spinal cord and optic nerve. Remarkably, though serum complement activity in rats is comparable to that in humans, no pathology was seen in peripheral AQP4-expressing organs or in circumventricular organs, such as area postrema in brain. The reason(s) why peripheral organs are spared in NMO are not clear; it has been speculated that the specialized environment in central nervous system tissues may be responsible, as might the differential expression of complement inhibitor proteins, such as CD55 and CD59, in central nervous system versus peripheral tissues [44‐47]. The absence of pathology in circumventricular organs in brains of seropositive rats suggests that the initiation of NMO pathogenesis requires some additional insult, perhaps local inflammation. Of note, humans may be seropositive for many years prior to clinical signs of NMO [48], and an inflammatory disease, such as gastroenteritis, is anecdotally reported to precede clinical NMO disease.

After evaluating several maneuvers to produce NMO pathology in seropositive rats, we found that a single needle insertion into brain parenchyma was sufficient to produce robust lesions around the needle track, with the characteristic pathological features of human NMO including loss of AQP4, GFAP and myelin, vasculocentric deposition of activated complement, granulocyte and macrophage infiltration, and blood–brain barrier disruption. Significant pathology was absent in rats administered non-NMO human IgG. The absence of significant pathology in similarly treated rats receiving NMO-IgG but made complement-deficient by cobra venom factor indicates the requirement for complement in this model. It should be informative to evaluate the roles of ADCC, neutrophils, eosinophils and macrophages in this model, as done previously in other NMO models [26, 16, 27‐29, 33], as well as to test therapeutics targeting complement [37, 38], NMO-IgG pathogenicity [35, 36], NMO-IgG binding to AQP4 [34, 49], and leukocyte-targeted drugs such as sivelestat [27] and cetirizine [29].

The NMO pathology seen at 5 days after intracerebral needle injury in seropositive rats was around the needle track in an area corresponding to that of early NMO-IgG diffusion. At one day astrocyte damage with loss of AQP4 and GFAP was seen, but little myelin loss. Interestingly, at one day multifocal punctate lesions were also seen away from the needle track that disappeared by five days. These lesions may represent mild, reversible astrocyte injury. A recent paper reported diffuse punctate lesions in ~60% of seropositive rats injected with IL-1β, but without lesions around the needle track [18]. In that study the level of NMO patient-derived IgG in rat serum was not measured, though it was likely to be quite low, as a single intraperitoneal injection of 10 mg of NMO patient-derived IgG in rat is much lower than 75 mg used in our study, which was chosen to produce serum cytotoxicity similar to that in human NMO. We speculate that the substantially lower serum NMO-IgG concentration in the study of Kitic et al. [18] was responsible for absence of robust NMO pathology.

A robust, passive-transfer model of NMO was established involving intracerebral needle stab injury in rats made seropositive by IP administration of NMO-IgG. The model does not require administration of complement or pro-inflammatory factors, or pre-existing inflammation. The model should be useful for further evaluation of NMO pathogenesis mechanisms and for evaluation of NMO therapeutics targeting circulating NMO-IgG, NMO-IgG binding to AQP4, complement, and inflammatory cells and factors.